Amino acid compositions and methods for the treatment of liver diseases

ABSTRACT

This disclosure provides pharmaceutical compositions comprising amino acid entities and uses thereof. Methods for improving liver function and for treating liver diseases comprising administering an effective amount of the compositions to a subject in need thereof are also disclosed.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/004,918 filed Jun. 11, 2018, which is a continuation of U.S.application Ser. No. 15/858,444 filed Dec. 29, 2017, which is acontinuation of U.S. application Ser. No. 15/847,289 filed Dec. 19,2017, which claims priority to U.S. Ser. No. 62/436,073 filed Dec. 19,2016, U.S. Ser. No. 62/443,205 filed Jan. 6, 2017, U.S. Ser. No.62/491,773 filed Apr. 28, 2017, U.S. Ser. No. 62/545,322 filed Aug. 14,2017, and U.S. Ser. No. 62/576,267 filed Oct. 24, 2017, the contents ofwhich are each incorporated herein by reference in their entireties.

BACKGROUND

Non-alcoholic fatty liver disease (NAFLD) is a disease characterized byfatty deposits in the liver due to causes other than alcohol. NAFLD isthe most prevalent liver disease in developed countries and affectsclose to 25% of the people in the United States. Non-alcoholicsteatohepatitis (NASH) is the most severe form of NAFLD, which can leadto inflammation of the liver, fibrosis, cirrhosis, chronic liverfailure, and hepatocellular carcinoma (HCC).

Currently, there are no approved therapies for treating NASH or NAFLD.Accordingly, there is an unmet need for new treatments in NAFLD andNASH.

SUMMARY

Disclosed herein, at least in part, is a composition including at leastfour different amino acid entities.

In some embodiments, the composition is capable of one, two, three,four, five, or six or all of:

a) decreasing or preventing liver fibrosis;

b) decreasing or preventing liver injury;

c) decreasing or preventing hepatocyte inflammation;

d) improving, e.g., increasing, glucose tolerance;

e) decreasing or preventing steatosis;

f) decreasing or preventing hepatocyte ballooning; or

g) improving gut function.

In some embodiments, the composition comprises a leucine (L)-amino acidentity, an arginine (R)-amino acid entity, a glutamine (Q)-amino acidentity; and an antioxidant or reactive oxygen species (ROS) scavenger(e.g., a N-acetylcysteine (NAC) entity, e.g., NAC). In some embodiments,at least one amino acid entity is not provided as a peptide of more than20 amino acid residues in length.

In some embodiments:

(i) an amino acid entity (e.g., at least one, two, or three of the aminoacid entities) of (a) is selected from Table 2; and/or

(ii) (A) one or both of the R-amino acid entity and the Q-amino acidentity are present at a higher amount (wt. %) than the L-amino acidentity, or (B) the composition further comprises a serine (S)-amino acidentity.

In any of the aspects and embodiments disclosed herein, the wt. ratio ofthe L-amino acid entity, the R-amino acid entity, the L-glutamine or asalt thereof, and the NAC or salt thereof is about 0.5 to 3:0.5 to 4:1to 4:0.1 to 2.5, e.g., the wt. ratio of the L-amino acid entity, theR-amino acid entity, the L-glutamine or a salt thereof, and the NAC orsalt thereof is about 1:1.5:2:0.15or about 1:1.5:2:0.3. In certainembodiments, the wt. ratio of the L-amino acid entity, the R-amino acidentity, the L-glutamine or a salt thereof, and the NAC or salt thereofis about 1+/−15%:1.5+/−15%:2+/−15%:0.15+/−15% or about1+/−15%:1.5+/−15%:2+/−15%:0.3+/−15%. In any of the aforesaid embodimentsin this paragraph, the wt. ratio of the L-amino acid entity, the R-aminoacid entity, the L-glutamine or a salt thereof, and the NAC or saltthereof is about 1:0.75:2:0.15 or about 1:0.75:2:0.3. In any of theaforesaid embodiments in this paragraph, the wt. ratio of the L-aminoacid entity, the R-amino acid entity, the L-glutamine or a salt thereof,and the NAC or salt thereof is about1+/−15%:0.75+/−15%:2+/−15%:0.15+/−15% or about1+/−15%:0.75+/−15%:2+/−15%:0.3+/−15%.

In any of the aspects and embodiments disclosed herein, the wt. ratio ofthe L-amino acid entity, the I-amino acid entity, the V-amino acidentity, the R-amino acid entity, the L-glutamine or salt thereof, andthe NAC or salt thereof is about 1:0.5:0.5:1.5:2:0.15 or about1:0.5:0.5:1.5:2:0.3.

In any of the aspects and embodiments disclosed herein, the compositionfurther comprises one or both of L-glycine and L-serine. In someembodiments, the composition comprises an L-amino acid entity, anI-amino acid entity, an V-amino acid entity, an R-amino acid entity, anL-glutamine or a salt thereof, an NAC or a salt thereof, and anL-glycine. In certain embodiments, the composition comprises an L-aminoacid entity, an I-amino acid entity, a V-amino acid entity, an R-aminoacid entity, an L-glutamine or a salt thereof, an NAC or a salt thereof,and an L-serine. In certain embodiments, the composition comprises anL-amino acid entity, an I-amino acid entity, an V-amino acid entity, anR-amino acid entity, an L-glutamine or a salt thereof, an NAC or a saltthereof, an L-glycine, and an L-serine. In any of the aforesaidembodiments in this paragraph, the wt. ratio of the L-amino acid entity,the I-amino acid entity, the V-amino acid entity, the R-amino acidentity, the L-glutamine or salt thereof, and the NAC or salt thereof isabout 1:0.5:0.5:1.5:2:0.15 or about 1:0.5:0.5:1.5:2:0.3. In any of theaforesaid embodiments in this paragraph, the wt. ratio of the L-aminoacid entity, the I-amino acid entity, the V-amino acid entity, theR-amino acid entity, the L-glutamine or salt thereof, and the NAC orsalt thereof is about1+/−15%:0.5+/−15%:0.5+/−15%:1.5+/−15%:2+/−15%:0.15+/−15% or about1+/−15%:0.5+/−15%:0.5+/−15%:1.5+/−15%:2+/−15%:0.3+/−15%.

In any of the aspects and embodiments disclosed herein, the compositioncomprises about 0.5 g to about 10 g of the L-amino acid entity, about0.25 g to about 5 g of the I-amino acid entity, about 0.25 g to about 5g of the V-amino acid entity, about 0.5 g to about 20 g of the R-aminoacid entity, about 1 g to about 20 g of the L-glutamine or a saltthereof, and about 0.1 g to about 5 g of the NAC or a salt thereof,e.g., the composition comprises about 1 g of the L-amino acid entity,about 0.5 g of the I-amino acid entity, about 0.5 g of V-amino acidentity, about 1.5 g of R-amino acid entity, about 2 g of L-glutamine ora salt thereof, and about 0.15 g or about 0.3 g of NAC or a saltthereof. In certain embodiments, the composition comprises about 0.15 gof NAC. In certain embodiments, the composition comprises about 0.3 g ofNAC. In embodiments, the composition comprises about 4 g of the L-aminoacid entity, about 2 g of the I-amino acid entity, about 1 g of V-aminoacid entity, about 3 g of R-amino acid entity, about 4 g of L-glutamineor a salt thereof, and about 0.9 g of NAC or a salt thereof.

In any of the aspects and embodiments disclosed herein, the compositioncomprises about 4 g of the L-amino acid entity, about 2 g of the I-aminoacid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acidentity, about 4 g of L-glutamine or a salt thereof, about 0.9 g of NACor a salt thereof, and about 6 g of L-serine or a salt thereof. Inembodiments, the composition comprises about 4 g of the L-amino acidentity, about 2 g of the I-amino acid entity, about 1 g of V-amino acidentity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or asalt thereof, about 0.9 g of NAC or a salt thereof, and about 6.67 g ofL-serine or a salt thereof. In embodiments, the composition comprisesabout 4 g of the L-amino acid entity, about 2 g of the I-amino acidentity, about 1 g of V-amino acid entity, about 3 g of R-amino acidentity, about 4 g of L-glutamine or a salt thereof, about 0.9 g of NACor a salt thereof, about 9 g of L-serine or a salt thereof, and about 9g of L-glycine or a salt thereof. In embodiments, the compositioncomprises about 4 g of the L-amino acid entity, about 2 g of the I-aminoacid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acidentity, about 4 g of L-glutamine or a salt thereof, about 0.9 g of NACor a salt thereof, about 3.33 g of L-serine or a salt thereof, and about3.33 g of L-glycine or a salt thereof.

In one aspect, the invention features a composition including free aminoacids, wherein the amino acids include arginine, glutamine,N-acetylcysteine, and a branched-chain amino acid chosen from one, two,or all of leucine, isoleucine, and valine.

In any of the aspects and embodiments disclosed herein, thebranched-chain amino acid is leucine, isoleucine, and valine.

In any of the aspects and embodiments disclosed herein, the wt ratio ofleucine, isoleucine, valine, arginine, glutamine, N-acetylcysteine is1:0.5:0.5:1.5:2:0.15. In certain embodiments, the wt ratio of leucine,isoleucine, valine, arginine, glutamine, N-acetylcysteine is1+/−15%:0.5+/−15%:0.5+/−15%:1.5+/−15%:2+/−15%:0.15+/−15%.

In any of the aspects and embodiments disclosed herein, a total weight(wt) of the amino acids is about 2 g to about 60 g. In some embodiments,the total wt of the amino acids is about 6 g, about 12 g, about 18 g,about 24 g, or about 48 g.

In any of the aspects and embodiments disclosed herein, the compositionincludes about 0.5 g to about 10 g of leucine, about 0.25 g to about 5 gof isoleucine, about 0.25 g to about 5 g of valine, about 1 g to about20 g of arginine, about 1 g to about 20 g of glutamine, and about 0.1 gto about 5 g of N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the compositionincludes about 1 g of leucine, about 0.5 g of isoleucine, about 0.5 g ofvaline, about 1.5 g of arginine, about 2 g of glutamine, and about 0.15g of N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the compositionincludes about 2 g of leucine, about 1 g of isoleucine, about 1 g ofvaline, about 3.0 g of arginine, about 4 g of glutamine, and about 0.3 gof N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the compositionincludes about 4 g of leucine, about 2 g of isoleucine, about 2 g ofvaline, about 6.0 g of arginine, about 8 g of glutamine, and about 0.6 gof N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the amino acidsinclude about 10 wt % to about 30 wt % leucine, about 5 wt % to about 15wt % isoleucine, about 5 wt % to about 15 wt % valine, about 15 wt % toabout 40 wt % arginine, about 20 wt % to about 50 wt % glutamine, andabout 1 wt % to about 8 wt % N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the amino acidsinclude about 16 wt % to about 18 wt % leucine, about 7 wt % to about 9wt % isoleucine, about 7 wt % to about 9 wt % valine, about 28 wt % toabout 32 wt % arginine, about 31 wt % to about 34 wt % glutamine, andabout 1 wt % to about 5 wt % N-acetylcysteine.

In any of the aspects and embodiments disclosed herein, the amino acidsinclude about 16.8 wt % leucine, about 8.4 wt % isoleucine, about 8.4 wt% valine, about 30.4 wt % arginine, about 33.6 wt % glutamine, and about2.5 wt % N-acetylcysteine.

In some embodiments of any of the compositions or methods disclosedherein (wherein the ratios discussed in (1)-(26) below are weightratios):

1) the ratio of the L-amino acid entity to the I-amino acid entity is atleast 1.5:1, or at least 1.75:1, and not more than 2.5:1 or not morethan 3:1, e.g., the ratio of the L-amino acid entity to the I-amino acidentity is about 2:1;

2) the ratio of L-amino acid entity to V-amino acid entity is at least2:1, at least 3:1, at least 3.5:1, at least 4:1, or at least 5:1, andnot more than 6:1, e.g., the ratio of L-amino acid entity to V-aminoacid entity is about 4:1;

3) the ratio of the L-amino acid entity to the R-amino acid entity is atleast 1:1, at least 3.5:3, at least 4:3, or at least 2:1, and not morethan 5:2, e.g., the ratio of the L-amino acid entity to the R-amino acidentity is about 4:3;

4) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is at least 0.5:1, or at least 0.75:1, and not more than 1.5 to1 or not more than 2:1, e.g., the ratio of the L-amino acid entity tothe L-glutamine or salt thereof is about 1:1;

5) the ratio of the L-amino acid entity to the NAC entity or a saltthereof is at least 2:1, at least 3:1, at least 3.5:1, or at least 4:1,and not more than 5 to 1 or not more than 6:1, e.g., the ratio of theL-amino acid entity to the NAC entity or salt thereof is about 4:1(e.g., 4:0.9);

6) optionally wherein the ratio of the L-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:3, greater than1.5:3, about 2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g.,the ratio of the L-amino acid entity to the S-amino acid entity is about2:3, or the ratio of the L-amino acid entity to the S-amino acid entityis about 3:5; or

7) a combination of two, three, four, five, or six of (1)-(6).

In some embodiments of any of the compositions or methods disclosedherein:

8) the ratio of I-amino acid entity to V-amino acid entity is at least1:1, at least 1.5:1, or about 2:1, and not more than 2.5:1 or not morethan 3:1, e.g., the ratio of I-amino acid entity to V-amino acid entityis about 2:1;

9) the ratio of the I-amino acid entity to the R-amino acid entity isgreater than 1:3, greater than 1.5:3, or about 2:3, and not more than2.5:3 or not more than 1:1, e.g., the ratio of the I-amino acid entityto the R-amino acid entity is about 2:3;

10) the ratio of the I-amino acid entity to the L-glutamine or a saltthereof is at least 1:4, at least 1:3, or about 1:2, and not more than1:1 or not more than 2:1, e.g., the ratio of the I-amino acid entity tothe L-glutamine or salt thereof is about 1:2;

11) the ratio of the I-amino acid entity to the NAC entity or a saltthereof is at least 1:1, at least 1.5:1, or about 2:1, and not more than2.5:1 or not more than 3:1, e.g., the ratio of the I-amino acid entityto the NAC entity or salt thereof is about 2:1 (e.g., 2:0.9);

12) optionally wherein the ratio of the I-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:4, greater than1.5:4, about 1:3, or about 3:10, and not more than 1.5:3 or 2:3, e.g.,the ratio of the I-amino acid entity to the S-amino acid entity is about1:3, or the ratio of the I-amino acid entity to the S-amino acid entityis about 3:10; or

13) a combination of two, three, four, or five of (8)-(12).

In some embodiments of any of the compositions or methods disclosedherein:

14) the ratio of the V-amino acid entity to the R-amino acid entity isgreater than 1:4, greater than 1.5:4, or about 1:3, and not more than1:2 or not more than 1:1, e.g., the ratio of the V-amino acid entity tothe R-amino acid entity is about 1:3;

15) the ratio of the V-amino acid entity to the L-glutamine or a saltthereof is greater than 1:5, or greater than 1.5:5, about 1:4, and notmore than 1.5:4 or not more than 1:3, e.g., the ratio of the V-aminoacid entity to the L-glutamine or salt thereof is about 1:4;

16) the ratio of the V-amino acid entity to the NAC entity or a saltthereof is at least 1:2, at least 1.5:2, or about 1:1, and not more than1.5:1 or not more than 2:1, e.g., the ratio of the V-amino acid entityto the NAC entity or salt thereof is about 1:1 (e.g., 1:0.9);

17) optionally wherein the ratio of the V-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:8, greater than1:7, about 1:6, or about 3:20, and not more than 1.5:6 or 1:3, e.g., theratio of the V-amino acid entity to the S-amino acid entity is about1:6, or the ratio of the V-amino acid entity to the S-amino acid entityis about 3:20; or

18) a combination of two, three, or four of (14)-(17).

In some embodiments of any of the compositions or methods disclosedherein:

19) the ratio of the R-amino acid entity to the L-glutamine or a saltthereof is greater than 1:2, greater than 1.25:2, or about 3:4, and notmore than 3.5:4 or not more than 1:1, e.g., the ratio of the R-aminoacid entity to the L-glutamine or salt thereof is about 3:4;

20) the ratio of the R-amino acid entity to the NAC entity or a saltthereof is at least 4:1, at least 4:1.5, or about 3:1, and not more than3:1.5 or not more than 3:2, e.g., the ratio of the R-amino acid entityto the NAC entity or salt thereof is about 3:1 (e.g., 3:0.9);

21) optionally wherein the ratio of the R-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:4, greater than1:3, about 1:2, or about 9:20, and not more than 1.5:2 or 1:1, e.g., theratio of the R-amino acid entity to the S-amino acid entity is about1:2, or the ratio of the R-amino acid entity to the S-amino acid entityis about 9:20; or

22) a combination of two or three of (19)-(21).

In some embodiments of any of the compositions or methods disclosedherein:

23) the ratio of the L-glutamine to the NAC entity or a salt thereof isat least 5:1, at least 5:1.5, or about 4:1, and not more than 4:1.5 ornot more than 3:1, e.g., the ratio of the L-glutamine to the NAC entityor salt thereof is about 4:1 (e.g., 4:0.9);

24) optionally wherein the ratio of the L-glutamine to the S-amino acidentity or a salt thereof is greater than 1:3, greater than 1.25:3, about2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g., the ratio ofthe L-glutamine to the S-amino acid entity is about 2:3, or the ratio ofthe L-glutamine to the S-amino acid entity is about 3:5; or

25) a combination of (23) and (24).

In some embodiments of any of the compositions or methods disclosedherein:

26) the ratio of the NAC entity to the S-amino acid entity or a saltthereof is greater than 1:8, greater than 1:7, or about 1:6, and notmore than 1:5 or not more than 1.5:5, e.g., the ratio of the NAC entityto the S-amino acid entity is about 1:6 (e.g., 0.9:6 or 2.7:20).

In an embodiment, the composition satisfies the properties of (1)-(7)defined above.

In certain embodiments, the composition satisfies the properties of atleast 2, 3, 4, 5, 6, or 7 of any of properties (1)-(26) defined above.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, and NAC or a salt thereof is12:6:3:9:12:2.7.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, andthe S-amino acid entity is 12:6:3:9:12:2.7:18.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, andthe S-amino acid entity is 12:6:3:9:12:2.7:20.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, and NAC or a salt thereof is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, andthe S-amino acid entity is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:18+/−15%.

In certain embodiments, the ratio of the L-amino acid-entity, theI-amino acid-entity, and the V-amino acid-entity in combination to theR-amino acid entity, L-glutamine or a salt thereof, NAC or a saltthereof, the S-amino acid entity, and the L-glycine is12:6:3:9:12:2.7:9:9. In certain embodiments, the ratio of the L-aminoacid-entity, the I-amino acid-entity, and the V-amino acid-entity incombination to the R-amino acid entity, L-glutamine or a salt thereof,NAC or a salt thereof, the S-amino acid entity, and the L-glycine is12:6:3:9:12:2.7:10:10.

In certain embodiments, the ratio of the L-amino acid-entity, theI-amino acid-entity, and the V-amino acid-entity in combination to theR-amino acid entity, L-glutamine or a salt thereof, NAC or a saltthereof, the S-amino acid entity, and the L-glycine is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:9+/−15%:9+/−15%. Incertain embodiments, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, theS-amino acid entity, and the L-glycine is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:10+/−15%:10+/−15%.

In any of the aspects and embodiments disclosed herein, the compositionfurther includes one or more pharmaceutically acceptable excipients.

In some embodiments, the excipients are selected from the groupconsisting of citric acid, lecithin, a sweetener, a dispersion enhancer,a flavoring, a bitterness masking agent, and a natural or artificialcoloring.

In some embodiments, the composition is in the form of a solid, powder,solution, or gel.

In some embodiments, the amino acids consist of leucine, isoleucine,valine, arginine, glutamine and N-acetylcysteine.

Another aspect of the invention features a dietary composition includingthe composition of any one of the foregoing aspects or embodiments,e.g., wherein the dietary composition is chosen from a medical food, afunctional food, or a supplement.

In some embodiments, the dietary composition is chosen from a medicalfood, a functional food, or a supplement.

In some embodiments, the subject has type 2 diabetes and/or a relativelyhigh BMI.

In some embodiments, the subject has non-alcoholic fatty liver disease(NAFLD).

In some embodiments, the subject has non-alcoholic fatty liver (NAFL).

In some embodiments, the subject has pediatric NAFLD.

In some embodiments, the patient has steatosis.

In some embodiments, the subject has non-alcoholic steatohepatitis(NASH).

In some embodiments, the subject has fibrosis.

In some embodiments, the subject has cirrhosis.

In some embodiments, the subject has AFLD.

In some embodiments, the subject has ASH.

In some embodiments, the subject has hepatocarcinoma, an increased riskof liver failure, or an increased risk of death.

In some embodiments, the subject has type 2 diabetes.

In some embodiments, the composition promotes weight loss in thesubject.

In some embodiments of the method or the dietary composition for use,the composition is administered at a dose of about 15 g/d to about 90g/d.

In some embodiments of the method or the dietary composition for use,the composition is administered at a dose of about 18 g/d, about 24 g/d,about 36/d, about 54 g/d, or about 72 g/d.

In some embodiments of the method or the dietary composition for use,the composition is administered one, two, to three times per day.

In some embodiments of the method or the dietary composition for use,the composition is administered at a dose of about 6 g, about 8 g, about12 g, about 16 g, about 18 g, or about 24 g three times per day.

One embodiment provides a nutritional supplement, dietary formulation,functional food, medical food, food, or beverage comprising acomposition described herein. Another embodiment provides a nutritionalsupplement, dietary formulation, functional food, medical food, food, orbeverage comprising a composition described herein for use in themanagement of any of the diseases or disorders described herein. Thecomposition disclosed herein can be used to improve liver function in asubject with fatty liver disease, such as non-alcoholic fatty liverdisease (NAFLD; e.g. NAFL or non-alcoholic steatohepatitis (NASH)) oralcoholic fatty liver disease (AFLD; e.g., alcoholic steatohepatitis(ASH)). Thus, a method, including a dosage regimen, for treating (e.g.,inhibiting, reducing, ameliorating, or preventing) various liverdisorders, diseases, or symptoms thereof using the amino acid entitycompositions is disclosed herein. The composition can also be used as adietary composition, e.g., a medical food, a functional food, or asupplement.

Another aspect of the invention features a method for treating one ormore symptoms selected from the group consisting of decreased fatmetabolism, hepatocyte apoptosis, hepatocyte ballooning, inflammation ofadipose tissue, inflammation of hepatic tissue, fibrosis, and oxidativestress, wherein the method includes administering to a subject in needthereof an effective amount of the composition of any one of aspects orembodiments disclosed herein.

In some embodiments, the subject has non-alcoholic fatty liver disease(NAFLD).

In some embodiments, the subject has non-alcoholic fatty liver (NAFL).

In some embodiments, the subject has pediatric NAFLD.

In some embodiments, the patient has steatosis.

In some embodiments, the subject has non-alcoholic steatohepatitis(NASH).

In some embodiments, the subject has alcoholic fatty liver disease(AFLD).

In some embodiments, the subject has alcoholic steatohepatitis (ASH)).

In some embodiments, the subject has fibrosis.

In some embodiments, the subject has cirrhosis.

In some embodiments, the subject has one, two, or more (e.g., all) ofhepatocarcinoma, an increased risk of liver failure, or an increasedrisk of death.

In some embodiments, the subject has type 2 diabetes.

Another aspect of the invention features a method for treatingnon-alcoholic fatty liver disease (NAFLD) including administering to asubject in need thereof an effective amount of the composition of anyone of the aspects or embodiments disclosed herein.

In some embodiments, the subject has NAFL.

In some embodiments, the subject has pediatric NAFLD.

In some embodiments, the patient has steatosis.

Another aspect of the invention features a method for treatingnon-alcoholic steatohepatitis (NASH) including administering to asubject in need thereof an effective amount of the composition of anyone of the aspects or embodiments disclosed herein.

In some embodiments, the subject has fibrosis.

Another aspect of the invention features a method for treating AFLDincluding administering to a subject in need thereof an effective amountof the composition of any one of the aspects or embodiments disclosedherein.

In some embodiments, the subject has ASH.

Another aspect of the invention features a method for treating cirrhosisincluding administering to a subject in need thereof an effective amountof the composition of any one of the aspects or embodiments disclosedherein.

In some embodiments, the subject has hepatocarcinoma, an increased riskof liver failure, or an increased risk of death.

In some embodiments, administering the composition results in animprovement in one or more metabolic symptoms in the subject. In someembodiments, the improvement in one or more metabolic symptoms isselected from the following: increased free fatty acid and lipidmetabolism, improved mitochondrial function, white adipose tissue (WAT)browning, decreased reactive oxygen species (ROS), increased levels ofglutathione (GSH), decreased hepatic inflammation, decreased hepatocyteballooning, improved gut barrier function, increased insulin secretion,or improved glucose tolerance.

In some embodiments, the increased free fatty acid and lipid metabolismoccurs in the liver.

In some embodiments, administration of the composition results in animprovement in one or more metabolic symptoms after a treatment periodof 24 hours.

In some embodiments, the method further includes determining the levelof one, two, three, four, five, six, seven, eight, nine, ten, or more(e.g., all) of the following:

a) alanine aminotransferase (ALT);

b) aspartate aminotransferase (AST);

c) adiponectin;

d) N-terminal fragment of type III collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

f) IL-1 beta;

g) C-reactive protein;

h) PIIINP;

i) a tissue inhibitor of metalloproteinase (TIMP); e.g., TIMP1 or TIMP2;

j) MCP-1;

k) FGF-21;

l) Col1a1;

m) Acta2;

n) a matrix metalloproteinase (MMP), e.g., MMP-13, MMP-2, MMP-9,MT1-MMP, MMP-3, or MMP-10;

o) ACOX1;

p) IL-10; or

q) NF-kB.

In some embodiments, administration of the composition results in animprovement in one or more of a)-q) after a treatment period of 24hours.

In some embodiments, the composition is administered prior to a meal.

In some embodiments, the composition is administered concurrent with ameal.

In some embodiments, the composition is administered following a meal.

In some embodiments, the composition is administered with a secondagent.

In some embodiments, the second agent is selected from the groupconsisting of a farnesoid X receptor (FXR) agonist, a stearoyl CoAdesaturase inhibitor, a CCR2 and CCR5 chemokine antagonist, a PPAR alphaand delta agonist, a caspase inhibitor, a galectin-3 inhibitor, anacetyl CoA carboxylase inhibitor, or an ileal sodium bile acidco-transporter inhibitor. Another aspect of the invention provides amethod of maintaining or improving liver health comprising administeringto a subject an effective amount of any of the compositions describedherein. Another embodiment provides a method of providing nutritionalsupport or supplementation to a subject suffering from NAFLD (e.g., NASHor NAFL) or AFLD (e.g., ASH) comprising administering to the subject aneffective amount of a composition described herein. Yet anotherembodiment provides a method of providing nutritional supplementationthat aids in the management of NAFLD (e.g., NASH or NAFL) or AFLD (e.g.,ASH) to a subject comprising administering to the subject in needthereof an effective amount of a composition described herein.

Additional features and embodiments of the present invention include oneor more of the following.

Another aspect of the invention features a composition comprising:

a) a L-amino acid entity chosen from L-leucine or a salt thereof, orβ-hydroxy-β-methybutyrate (HMB) or a salt thereof, or a combination ofL-leucine or a salt thereof and HMB or a salt thereof;

b) an R-amino acid entity chosen from: L-arginine or a salt thereof,ornithine or a salt thereof, or creatine or a salt thereof, or acombination of two or three of L-arginine or a salt thereof, ornithineor a salt thereof, or creatine or a salt thereof;

c) L-glutamine or a salt thereof; and

d) N-acetylcysteine (NAC) or a salt thereof.

In an embodiment, L-leucine is provided as part of a dipeptidecomprising L-leucine, or a salt thereof, or a tripeptide comprisingL-leucine, or a salt thereof.

In an embodiment, L-arginine is provided as part of a dipeptidecomprising L-arginine, or a salt thereof, or a tripeptide comprisingL-arginine, or a salt thereof.

In an embodiment L-glutamine is provided as part of a dipeptidecomprising L-glutamine, or a salt thereof, or a tripeptide comprisingL-glutamine, or a salt thereof.

In an embodiment NAC is provided as a part of a dipeptide comprisingNAC, or a salt thereof, or a tripeptide comprising NAC, or a saltthereof.

In some embodiments of any of the compositions or methods disclosedherein, one, two, three, or four of methionine (M), tryptophan (W),valine (V), or cysteine (C) is absent, or if present, is present at apercentage of the composition by weight (wt. %) of less than 10%. Insome embodiments, the total wt. % of (a)-(d) is greater than the totalwt. % of any other amino acid entity in the composition.

In some embodiments of any of the compositions or methods disclosedherein, one, two, three, or four of the amino acids in (a)-(d) isprovided as part of a dipeptide or tripeptide, e.g., in an amount of atleast 10 wt. % of the composition. In certain embodiments, the dipeptideis a homodipeptide or heterodipeptide of any of the amino acids in(a)-(d), e.g., one, two, three, or four of the amino acids in (a)-(d) isa homodipeptide or heterodipeptide. In certain embodiments, thetripeptide is a homotripeptide or heterotripeptide of any of (a)-(d),e.g., one, two, three, or four of (a)-(d) is a homotripeptide orheterotripeptide.

In some embodiments of any of the compositions or methods disclosedherein, (a) is a L-amino acid entity dipeptide or a salt thereof (e.g.,a L-leucine dipeptide or a salt thereof). In some embodiments, (a) is ahomodipeptide. In some embodiments, (a) is a heterodipeptide, e.g.,Ala-Leu.

In some embodiments of any of the compositions or methods disclosedherein, (b) is a L-arginine dipeptide or a salt thereof. In someembodiments, (b) is a homodipeptide. In some embodiments, (b) is aheterodipeptide, e.g., Ala-Arg.

In some embodiments of any of the compositions or methods disclosedherein, (c) is a L-glutamine dipeptide or a salt thereof. In someembodiments, (c) is a homodipeptide, e.g., Gln-Gln. In some embodiments,(c) is a heterodipeptide, e.g., Ala-Gln.

In some embodiments of any of the compositions or methods disclosedherein:

f) a wt. % of the L-glutamine or a salt thereof in the composition isgreater than the wt. % of the R-amino acid entity;

g) the wt. % of the L-glutamine or a salt thereof in the composition isgreater than the wt. % of the L-amino acid entity;

h) the wt. % of the R-amino acid entity in the composition is greaterthan the wt. % of the L-amino acid entity; or

i) a combination of two or three of (f)-(h).

In some embodiments of any of the compositions or methods disclosedherein, the wt. % of the L-glutamine or a salt thereof in thecomposition is at least 5% greater than the wt. % of the R-amino acidentity, e.g., the wt. % of the L-glutamine or a salt thereof is at least10%, 15%, 20%, or 25% greater than the wt. % of the R-amino acid entity.

In some embodiments of any of the compositions or methods disclosedherein, the wt. % of the L-glutamine or a salt thereof in thecomposition is at least 20% greater than the wt. % of the L-amino acidentity, e.g., the wt. % of the L-glutamine or a salt thereof in thecomposition is at least 25%, 30%, 35%, 40%, 45%, or 50% greater than thewt. % of the L-amino acid entity.

In some embodiments of any of the compositions or methods disclosedherein, the wt. % of the R-amino acid entity in the composition is atleast 10% greater than the wt. % of the L-amino acid entity, e.g., thewt. % of the R-amino acid entity in the composition is at least 15%,20%, 25%, or 30% greater than the wt. % of the L-amino acid entity.

In some embodiments of any of the compositions or methods disclosedherein:

j) the ratio of the L-amino acid entity to the R-amino acid entity is atleast 1:4, or at least 2:5, and not more than 3:4, e.g., the ratio ofL-amino acid entity to R-amino acid entity is about 2:3;

k) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is at least 1:4, or at least 1:3, and not more than 3:4, e.g.,the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is about 1:2;

l) the ratio of the R-amino acid entity to the L-glutamine or a saltthereof is at least 1:4, or at least 1:2, and not more than 6:7, e.g.,the ratio of the R-amino acid entity to the L-glutamine or a saltthereof is about 3:4; or

m) a combination of two or three of (j)-(l).

In an embodiment, the composition satisfies the properties of (j)-(l)defined above.

In certain embodiments, the composition satisfies the properties of atleast 2, 3, or 4 of any of properties (j)-(m) defined above.

In some embodiments of any of the compositions or methods disclosedherein, the composition further comprises one or both of an isoleucine(I)-amino acid-entity and a valine (V)-amino acid-entity, e.g., both theI-amino acid-entity and the V-amino acid-entity are present.

In some embodiments of any of the compositions or methods disclosedherein:

n) the wt. % of the L-amino acid-entity in the composition is greaterthan or equal to the wt. % of the I-amino acid-entity and the V-aminoacid-entity in combination;

o) the wt. % of the L-amino acid-entity, the I-amino acid-entity, andthe V-amino acid-entity in combination in the composition is greaterthan or equal to the wt. % of the L-glutamine or a salt thereof;

p) the wt. % of the L-amino acid-entity, the I-amino acid-entity, andthe V-amino acid-entity in combination in the composition is less thanthe wt. % of the R-amino acid entity;

q) the wt. % of the R-amino acid entity and the L-glutamine or a saltthereof in the composition is greater than the wt. % of the L-aminoacid-entity, the I-amino acid-entity, and the V-amino acid-entity incombination; or

r) a combination of two, three, or four of (n)-(q).

In some embodiments of any of the compositions or methods disclosedherein:

s) the wt. % of the R-amino acid entity, the L-glutamine or a saltthereof, and the NAC or a salt thereof is at least 50% of thecomposition, or at least 70% of the composition, but not more than 90%of the composition;

t) the wt. % of the NAC or a salt thereof is at least 1%, or at least2%, but not more than 10% of the composition;

u) the wt. % of the L-amino acid-entity, the I-amino acid-entity, andthe V-amino acid-entity in combination is at least 15%, or at least 20%,but not more than 50% of the composition;

v) the wt. % of the R-amino acid entity, the L-glutamine or a saltthereof, and the NAC or a salt thereof is at least 40%, or at least 50%,but not more than 80% of the composition; or

w) a combination of two, three, or four of (s)-(v).

In some embodiments of any of the compositions or methods disclosedherein:

x) the ratio of the L-amino acid entity to the I-amino acid entity is atleast 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not morethan 3:1, e.g., the ratio of the L-amino acid entity to the I-amino acidentity is about 2:1;

y) the ratio of L-amino acid entity to V-amino acid entity is at least1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not more than3:1, e.g., the ratio of L to V is about 2:1;

z) the ratio of the L-amino acid entity to the R-amino acid entity isgreater than 1:3, greater than 1.5 to 3, and less than 3:3, e.g., theratio of the L-amino acid entity to the R-amino acid entity is about2:3;

aa) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is greater than 1:4, greater than 1.5 to 4 and less than 4:4, orless than 3:4, e.g., the ratio of the L-amino acid entity to theL-glutamine or a salt thereof is about 1:2; or

bb) a combination of two, three, or four of (x)-(aa).

In an embodiment, the composition satisfies the properties of (x)-(aa)defined above.

In certain embodiments, the composition satisfies the properties of atleast 2, 3, 4, or 5 of any of properties (x)-(bb) defined above.

In some embodiments of any of the compositions or methods disclosedherein:

cc) the ratio of the I-amino acid entity to the V-amino acid entity isat least 0.5:1, or at least 0.75:1, and not more than 1.5 to 1 or notmore than 2:1, e.g., the ratio of the L-amino acid entity to the I-aminoacid entity is about 1:1;

dd) the ratio of the I-amino acid entity to the R-amino acid entity isat least 0.5:3, or at least 0.75:3, and not more than 2:3, or not morethan 1.5:3, e.g., the ratio of the L-amino acid entity to the I-aminoacid entity is about 1:3;

ee) the ratio of the I-amino acid entity to the L-glutamine or a saltthereof is at least 0.5:4, or at least 0.75:4, and not more than 3:4, ornot more than 2:4, e.g., the ratio of the L-amino acid entity to theL-glutamine or a salt thereof is about 1:4; or

ff) or a combination of two or three of (cc)-(ee).

In an embodiment, the composition satisfies the properties of (cc)-(ee)defined above.

In certain embodiments, the composition satisfies the properties of atleast 2, 3, or 4 of any of properties (cc)-(ff) defined above.

In some embodiments of any of the compositions or methods disclosedherein:

gg) the ratio of the L-amino acid entity to the V-amino acid entity isat least 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or notmore than 3:1, e.g., the ratio of the L-amino acid entity to the V-aminoacid entity is about 2:1;

hh) the ratio of the L-amino acid entity to the R-amino acid entity isgreater than 1:3 or greater than 1.5 to 3, and less than 3:3, e.g., theratio of the L-amino acid entity to the R-amino acid entity is about2:3;

ii) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is greater than 1:4 or greater than 1.5 to 4, and less than 4:4or less than 3:4, e.g., the ratio of the L-amino acid entity to theL-glutamine or a salt thereof is about 1:2; or

jj) a combination of two or three of (gg)-(ii).

In an embodiment, the composition satisfies the properties of (gg)-(ii)defined above.

In certain embodiments, the composition satisfies the properties of atleast 2, 3, or 4 of any of properties (gg)-(jj) defined above.

In some embodiments of any of the compositions or methods disclosedherein:

kk) the ratio of the V-amino acid entity to the L-glutamine or a saltthereof is at least 0.5:4, or at least 0.75:4, and not more than 3:4, ornot more than 2:4, e.g., the ratio of the L-amino acid entity to theL-glutamine or a salt thereof is about 1:4;

ll) the ratio of the V-amino acid entity to the R-amino acid entity isat least 0.5:3, or at least 0.75:3, and not more than 2:3, or not morethan 1.5:3, e.g., the ratio of the V-amino acid entity to the R-aminoacid entity is about 1:3;

mm) the ratio of the L-amino acid-entity, the I-amino acid-entity, andthe V-amino acid-entity in combination to the R-amino acid entity,L-glutamine or a salt thereof, and NAC or a salt thereof is at least1:4, or at least 2:3, or not more than 5:7, or not more than 6:7, e.g.,the ratio is about 6:11; or

nn) a combination of two or three of (kk)-(mm).

In an embodiment, the composition satisfies the properties of (kk)-(mm)defined above.

In certain embodiments, the composition satisfies the properties of atleast 2, 3, or 4 of any of properties (kk)-(nn) defined above.

In some embodiments of any of the compositions or methods disclosedherein, the composition further comprises an S-amino acid entity.

In some embodiments of any of the compositions or methods disclosedherein:

1) the ratio of the L-amino acid entity to the I-amino acid entity is atleast 1.5:1, or at least 1.75:1, and not more than 2.5:1 or not morethan 3:1, e.g., the ratio of the L-amino acid entity to the I-amino acidentity is about 2:1;

2) the ratio of L-amino acid entity to V-amino acid entity is at least2:1, at least 3:1, at least 3.5:1, at least 4:1, or at least 5:1, andnot more than 6:1, e.g., the ratio of L-amino acid entity to V-aminoacid entity is about 4:1;

3) the ratio of the L-amino acid entity to the R-amino acid entity is atleast 1:1, at least 3.5:3, at least 4:3, or at least 2:1, and not morethan 5:2, e.g., the ratio of the L-amino acid entity to the R-amino acidentity is about 4:3;

4) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is at least 0.5:1, or at least 0.75:1, and not more than 1.5 to1 or not more than 2:1, e.g., the ratio of the L-amino acid entity tothe L-glutamine or salt thereof is about 1:1;

5) the ratio of the L-amino acid entity to the NAC entity or a saltthereof is at least 2:1, at least 3:1, at least 3.5:1, or at least 4:1,and not more than 5 to 1 or not more than 6:1, e.g., the ratio of theL-amino acid entity to the NAC entity or salt thereof is about 4:1(e.g., 4:0.9);

6) optionally wherein the ratio of the L-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:3, greater than1.5:3, about 2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g.,the ratio of the L-amino acid entity to the S-amino acid entity is about2:3, or the ratio of the L-amino acid entity to the S-amino acid entityis about 3:5; or

7) a combination of two, three, four, five, or six of (1)-(6).

In some embodiments of any of the compositions or methods disclosedherein:

8) the ratio of I-amino acid entity to V-amino acid entity is at least1:1, at least 1.5:1, or about 2:1, and not more than 2.5:1 or not morethan 3:1, e.g., the ratio of I-amino acid entity to V-amino acid entityis about 2:1;

9) the ratio of the I-amino acid entity to the R-amino acid entity isgreater than 1:3, greater than 1.5:3, or about 2:3, and not more than2.5:3 or not more than 1:1, e.g., the ratio of the I-amino acid entityto the R-amino acid entity is about 2:3;

10) the ratio of the I-amino acid entity to the L-glutamine or a saltthereof is at least 1:4, at least 1:3, or about 1:2, and not more than1:1 or not more than 2:1, e.g., the ratio of the I-amino acid entity tothe L-glutamine or salt thereof is about 1:2;

11) the ratio of the I-amino acid entity to the NAC entity or a saltthereof is at least 1:1, at least 1.5:1, or about 2:1, and not more than2.5:1 or not more than 3:1, e.g., the ratio of the I-amino acid entityto the NAC entity or salt thereof is about 2:1 (e.g., 2:0.9);

12) optionally wherein the ratio of the I-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:4, greater than1.5:4, about 1:3, or about 3:10, and not more than 1.5:3 or 2:3, e.g.,the ratio of the I-amino acid entity to the S-amino acid entity is about1:3, or the ratio of the I-amino acid entity to the S-amino acid entityis about 3:10; or

13) a combination of two, three, four, or five of (8)-(12).

In some embodiments of any of the compositions or methods disclosedherein:

14) the ratio of the V-amino acid entity to the R-amino acid entity isgreater than 1:4, greater than 1.5:4, or about 1:3, and not more than1:2 or not more than 1:1, e.g., the ratio of the V-amino acid entity tothe R-amino acid entity is about 1:3;

15) the ratio of the V-amino acid entity to the L-glutamine or a saltthereof is greater than 1:5, or greater than 1.5:5, about 1:4, and notmore than 1.5:4 or not more than 1:3, e.g., the ratio of the V-aminoacid entity to the L-glutamine or salt thereof is about 1:4;

16) the ratio of the V-amino acid entity to the NAC entity or a saltthereof is at least 1:2, at least 1.5:2, or about 1:1, and not more than1.5:1 or not more than 2:1, e.g., the ratio of the V-amino acid entityto the NAC entity or salt thereof is about 1:1 (e.g., 1:0.9);

17) optionally wherein the ratio of the V-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:8, greater than1:7, about 1:6, or about 3:20, and not more than 1.5:6 or 1:3, e.g., theratio of the V-amino acid entity to the S-amino acid entity is about1:6, or the ratio of the V-amino acid entity to the S-amino acid entityis about 3:20; or

18) a combination of two, three, or four of (14)-(17).

In some embodiments of any of the compositions or methods disclosedherein:

19) the ratio of the R-amino acid entity to the L-glutamine or a saltthereof is greater than 1:2, greater than 1.25:2, or about 3:4, and notmore than 3.5:4 or not more than 1:1, e.g., the ratio of the R-aminoacid entity to the L-glutamine or salt thereof is about 3:4;

20) the ratio of the R-amino acid entity to the NAC entity or a saltthereof is at least 4:1, at least 4:1.5, or about 3:1, and not more than3:1.5 or not more than 3:2, e.g., the ratio of the R-amino acid entityto the NAC entity or salt thereof is about 3:1 (e.g., 3:0.9);

21) optionally wherein the ratio of the R-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:4, greater than1:3, about 1:2, or about 9:20, and not more than 1.5:2 or 1:1, e.g., theratio of the R-amino acid entity to the S-amino acid entity is about1:2, or the ratio of the R-amino acid entity to the S-amino acid entityis about 9:20; or

22) a combination of two or three of (19)-(21).

In some embodiments of any of the compositions or methods disclosedherein:

23) the ratio of the L-glutamine to the NAC entity or a salt thereof isat least 5:1, at least 5:1.5, or about 4:1, and not more than 4:1.5 ornot more than 3:1, e.g., the ratio of the L-glutamine to the NAC entityor salt thereof is about 4:1 (e.g., 4:0.9);

24) optionally wherein the ratio of the L-glutamine to the S-amino acidentity or a salt thereof is greater than 1:3, greater than 1.25:3, about2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g., the ratio ofthe L-glutamine to the S-amino acid entity is about 2:3, or the ratio ofthe L-glutamine to the S-amino acid entity is about 3:5; or

25) a combination of (23) and (24).

In some embodiments of any of the compositions or methods disclosedherein:

26) the ratio of the NAC entity to the S-amino acid entity or a saltthereof is greater than 1:8, greater than 1:7, or about 1:6, and notmore than 1:5 or not more than 1.5:5, e.g., the ratio of the NAC entityto the S-amino acid entity is about 1:6 (e.g., 0.9:6 or 2.7:20).

In an embodiment, the composition satisfies the properties of (1)-(7)defined above.

In certain embodiments, the composition satisfies the properties of atleast 2, 3, 4, 5, 6, or 7 of any of properties (1)-(26) defined above.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, and NAC or a salt thereof is12:6:3:9:12:2.7.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, andthe S-amino acid entity is 12:6:3:9:12:2.7:18.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, andthe S-amino acid entity is 12:6:3:9:12:2.7:20.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, and NAC or a salt thereof is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%.

In an embodiment, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, andthe S-amino acid entity is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:18+/−15%.

In certain embodiments, the ratio of the L-amino acid-entity, theI-amino acid-entity, and the V-amino acid-entity in combination to theR-amino acid entity, L-glutamine or a salt thereof, NAC or a saltthereof, the S-amino acid entity, and the L-glycine is12:6:3:9:12:2.7:9:9. In certain embodiments, the ratio of the L-aminoacid-entity, the I-amino acid-entity, and the V-amino acid-entity incombination to the R-amino acid entity, L-glutamine or a salt thereof,NAC or a salt thereof, the S-amino acid entity, and the L-glycine is12:6:3:9:12:2.7:10:10.

In certain embodiments, the ratio of the L-amino acid-entity, theI-amino acid-entity, and the V-amino acid-entity in combination to theR-amino acid entity, L-glutamine or a salt thereof, NAC or a saltthereof, the S-amino acid entity, and the L-glycine is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:9+/−15%:9+/−15%. Incertain embodiments, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, theS-amino acid entity, and the L-glycine is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:10+/−15%:10+/−15%.

In some embodiments of any of the compositions or methods disclosedherein:

oo) a wt. % of the L-amino acid entity in the composition is greaterthan the wt. % of the NAC or a salt thereof;

pp) a wt. % of the R-amino acid entity in the composition is greaterthan the wt. % of the NAC or a salt thereof;

qq) a wt. % of the L-glutamine or a salt thereof in the composition isgreater than the wt. % of the NAC or a salt thereof; or

rr) a combination of two or three of (oo)-(qq).

In some embodiments of any of the compositions or methods disclosedherein, at least one of (a)-(d) is a free amino acid, e.g., two, three,or four of (a)-(d) are a free amino acid, e.g., at least 50 wt. % of thetotal wt. of the composition is one or more amino acid entities in freeform.

In some embodiments of any of the compositions or methods disclosedherein, at least one of (a)-(d) is in a salt form, e.g., one, two,three, or four of (a)-(d) is in a salt form, e.g., at least 10 wt. % ofthe total wt. of the composition is one or more amino acid entities insalt form.

In some embodiments of any of the compositions or methods disclosedherein, the composition is capable of one, two, three, four, five, orall of:

a) decreasing or preventing liver fibrosis;

b) decreasing or preventing liver injury;

c) decreasing or preventing hepatocyte inflammation;

d) improving, e.g., increasing, glucose tolerance;

e) decreasing or preventing steatosis;

f) decreasing or preventing hepatocyte ballooning; or

g) improving gut function.

In some embodiments of any of the compositions or methods disclosedherein, the composition further comprises one or both of L-glycine andL-serine. In certain embodiments, the composition comprises an L-aminoacid entity, an I-amino acid entity, an V-amino acid entity, an R-aminoacid entity, an L-glutamine or a salt thereof, an NAC or a salt thereof,and an L-glycine. In certain embodiments, the composition comprises anL-amino acid entity, an I-amino acid entity, an V-amino acid entity, anR-amino acid entity, an L-glutamine or a salt thereof, an NAC or a saltthereof, and an L-serine. In certain embodiments, the compositioncomprises an L-amino acid entity, an I-amino acid entity, an V-aminoacid entity, an R-amino acid entity, an L-glutamine or a salt thereof,an NAC or a salt thereof, an L-glycine, and an L-serine. In any of theaforesaid embodiments in this paragraph, the wt. ratio of the L-aminoacid entity, the I-amino acid entity, the V-amino acid entity, theR-amino acid entity, the L-glutamine or salt thereof, and the NAC orsalt thereof is about 1:0.5:0.5:1.5:2:0.15 or about 1:0.5:0.5:1.5:2:0.3.

In some embodiments of any of the compositions or methods disclosedherein, the wt. ratio of the L-amino acid entity, the R-amino acidentity, the L-glutamine or a salt thereof, and the NAC or salt thereofis about 0.5 to 3:0.5 to 4:1 to 4:0.1 to 2.5, e.g., the wt. ratio of theL-amino acid entity, the R-amino acid entity, the L-glutamine or a saltthereof, and the NAC or salt thereof is about 1:1.5:2:0.15, about1:1.5:2:0.225, about 1:1.5:2:0.3, or about 1:1.5:2:0.5. In any of theaforesaid embodiments in this paragraph, the wt. ratio of the L-aminoacid entity, the R-amino acid entity, the L-glutamine or a salt thereof,and the NAC or salt thereof is about 1:0.75:2:0.15, about1:0.75:2:0.225, about 1:0.75:2:0.3, or about 1:0.75:2:0.5.

In some embodiments, the wt. ratio of the L-amino acid entity, theI-amino acid entity, the V-amino acid entity, the R-amino acid entity,the L-glutamine or salt thereof, and the NAC or salt thereof is about1:0.5:0.5:1.5:2:0.15, about 1:0.5:0.5:1.5:2:0.225, about1:0.5:0.5:1.5:2:0.3, or about 1:0.5:0.5:1.5:2:0.5.

In some embodiments of any of the compositions or methods disclosedherein, the composition comprises about 0.5 g to about 10 g of theL-amino acid entity, about 0.25 g to about 5 g of the I-amino acidentity, about 0.25 g to about 5 g of the V-amino acid entity, about 0.5g to about 20 g of the R-amino acid entity, about 1 g to about 20 g ofthe L-glutamine or a salt thereof, and about 0.1 g to about 5 g of theNAC or a salt thereof, e.g., the composition comprises about 1 g of theL-amino acid entity, about 0.5 g of the I-amino acid entity, about 0.5 gof V-amino acid entity, about 1.5 g of R-amino acid entity, about 2 g ofL-glutamine or a salt thereof, and about 0.15 g, about 0.225 g, about0.3 g, or about 0.5 g of NAC or a salt thereof. In certain embodiments,the composition comprises about 0.15 g of NAC. In certain embodiments,the composition comprises about 0.3 g of NAC. In embodiments, thecomposition comprises about 4 g of the L-amino acid entity, about 2 g ofthe I-amino acid entity, about 1 g of V-amino acid entity, about 3 g ofR-amino acid entity, about 4 g of L-glutamine or a salt thereof, andabout 0.9 g of NAC or a salt thereof. In embodiments, the compositioncomprises about 4 g of the L-amino acid entity, about 2 g of the I-aminoacid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acidentity, about 4 g of L-glutamine or a salt thereof, about 0.9 g of NACor a salt thereof, and about 6 g of L-serine or a salt thereof. Inembodiments, the composition comprises about 4 g of the L-amino acidentity, about 2 g of the I-amino acid entity, about 1 g of V-amino acidentity, about 3 g of R-amino acid entity, about 4 g of L-glutamine or asalt thereof, about 0.9 g of NAC or a salt thereof, and about 6.67 g ofL-serine or a salt thereof. In embodiments, the composition comprisesabout 4 g of the L-amino acid entity, about 2 g of the I-amino acidentity, about 1 g of V-amino acid entity, about 3 g of R-amino acidentity, about 4 g of L-glutamine or a salt thereof, about 0.9 g of NACor a salt thereof, about 3 g of L-serine or a salt thereof, and about 3g of L-glycine or a salt thereof. In embodiments, the compositioncomprises about 4 g of the L-amino acid entity, about 2 g of the I-aminoacid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acidentity, about 4 g of L-glutamine or a salt thereof, about 0.9 g of NACor a salt thereof, about 3.33 g of L-serine or a salt thereof, and about3.33 g of L-glycine or a salt thereof.

In some embodiments of any of the compositions or methods disclosedherein, the composition comprises:

-   -   a) L-Leucine or a salt thereof;    -   b) L-Isoleucine or a salt thereof;    -   c) L-Valine or a salt thereof;    -   d) L-Arginine or a salt thereof;    -   e) L-Glutamine or a salt thereof; and    -   f) NAC or a salt thereof.

In an embodiment, L-Leucine is provided as part of a dipeptidecomprising L-Leucine, or a salt thereof, or a tripeptide comprisingL-Leucine, or a salt thereof.

In an embodiment, L-Isoleucine is provided as part of a dipeptidecomprising L-Isoleucine, or a salt thereof, or a tripeptide comprisingL-Isoleucine, or a salt thereof.

In an embodiment, L-Valine is provided as part of a dipeptide comprisingL-Valine, or a salt thereof, or a tripeptide comprising L-Valine, or asalt thereof.

In an embodiment, L-Arginine is provided as part of a dipeptidecomprising L-Arginine, or a salt thereof, or a tripeptide comprisingL-Arginine, or a salt thereof.

In an embodiment L-Glutamine is provided as part of a dipeptidecomprising L-Glutamine, or a salt thereof, or a tripeptide comprisingL-Glutamine, or a salt thereof.

In an embodiment NAC is provided as a part of a dipeptide comprisingNAC, or a salt thereof, or a tripeptide comprising NAC, or a saltthereof.

In some embodiments of any of the compositions or methods disclosedherein, the composition comprises a combination of 4 to 20 differentamino acid entities, e.g., a combination of 5 to 15 different amino acidentities.

In some embodiments of any of the compositions or methods disclosedherein, at least two, three, four, or more amino acid entities are notcomprised in a peptide of more than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, or 20 amino acid residues in length.

Another aspect of the invention features a method for improving liverfunction, wherein the method comprises administering to a subject inneed thereof an effective amount of a composition comprising:

a) a L-amino acid entity chosen from L-leucine or a salt thereof, orβ-hydroxy-β-methybutyrate (HMB) or a salt thereof, or a combination ofL-leucine or a salt thereof and HMB or a salt thereof;

b) an R-amino acid entity chosen from: L-arginine or a salt thereof,ornithine or a salt thereof, or creatine or a salt thereof, or acombination of two or three of L-arginine or a salt thereof, ornithineor a salt thereof, or creatine or a salt thereof;

c) L-glutamine or a salt thereof; and

d) N-acetylcysteine (NAC) or a salt thereof.

In an embodiment, L-Leucine is provided as part of a dipeptidecomprising L-Leucine, or a salt thereof, or a tripeptide comprisingL-Leucine, or a salt thereof.

In an embodiment, L-Arginine is provided as part of a dipeptidecomprising L-Arginine, or a salt thereof, or a tripeptide comprisingL-Arginine, or a salt thereof.

In an embodiment L-Glutamine is provided as part of a dipeptidecomprising L-Glutamine, or a salt thereof, or a tripeptide comprisingL-Glutamine, or a salt thereof.

In an embodiment NAC is provided as a part of a dipeptide comprisingNAC, or a salt thereof, or a tripeptide comprising NAC, or a saltthereof.

Another aspect of the invention features a method for treating one ormore symptoms selected from the group consisting of decreased fatmetabolism, hepatocyte apoptosis, hepatocyte ballooning, inflammation ofadipose tissue, inflammation of hepatic tissue, fibrosis, liver injury,steatosis, glucose tolerance, and oxidative stress, wherein the methodcomprises administering to a subject in need thereof an effective amountof a composition comprising:

a) a L-amino acid entity chosen from L-leucine or a salt thereof, orβ-hydroxy-β-methybutyrate (HMB) or a salt thereof, or a combination ofL-leucine or a salt thereof and HMB or a salt thereof;

b) an R-amino acid entity chosen from: L-arginine or a salt thereof,ornithine or a salt thereof, or creatine or a salt thereof, or acombination of two or three of L-arginine or a salt thereof, ornithineor a salt thereof, or creatine or a salt thereof;

c) L-glutamine or a salt thereof; and

d) N-acetylcysteine (NAC) or a salt thereof.

In an embodiment, L-Leucine is provided as part of a dipeptidecomprising L-Leucine, or a salt thereof, or a tripeptide comprisingL-Leucine, or a salt thereof.

In an embodiment, L-Arginine is provided as part of a dipeptidecomprising L-Arginine, or a salt thereof, or a tripeptide comprisingL-Arginine, or a salt thereof.

In an embodiment L-Glutamine is provided as part of a dipeptidecomprising L-Glutamine, or a salt thereof, or a tripeptide comprisingL-Glutamine, or a salt thereof.

In an embodiment NAC is provided as a part of a dipeptide comprisingNAC, or a salt thereof, or a tripeptide comprising NAC, or a saltthereof.

Another aspect of the invention features a method for treating fattyliver disease, wherein the method comprises administering to a subjectin need thereof an effective amount of a composition comprising:

a) a L-amino acid entity chosen from L-leucine or a salt thereof, orβ-hydroxy-β-methylbutyrate (HMB) or a salt thereof, or a combination ofL-leucine or a salt thereof and HMB or a salt thereof;

b) an R-amino acid entity chosen from: L-arginine or a salt thereof,ornithine or a salt thereof, or creatine or a salt thereof, or acombination of two or three of L-arginine or a salt thereof, ornithineor a salt thereof, or creatine or a salt thereof;

c) L-glutamine or a salt thereof; and

d) N-acetylcysteine (NAC) or a salt thereof.

In an embodiment, L-Leucine is provided as part of a dipeptidecomprising L-Leucine, or a salt thereof, or a tripeptide comprisingL-Leucine, or a salt thereof.

In an embodiment, L-Arginine is provided as part of a dipeptidecomprising L-Arginine, or a salt thereof, or a tripeptide comprisingL-Arginine, or a salt thereof.

In an embodiment L-Glutamine is provided as part of a dipeptidecomprising L-Glutamine, or a salt thereof, or a tripeptide comprisingL-Glutamine, or a salt thereof.

In an embodiment NAC is provided as a part of a dipeptide comprisingNAC, or a salt thereof, or a tripeptide comprising NAC, or a saltthereof.

In some embodiments of any of the compositions or methods disclosedherein, e.g., of any of the methods described herein, the subject has adisease or disorder selected from the group consisting of non-alcoholicfatty liver (NAFL), non-alcoholic fatty liver disease (NAFLD),non-alcoholic steatohepatitis (NASH), alcoholic fatty liver disease(AFLD), and alcoholic steatohepatitis (ASH). In certain embodiments, thesubject has pediatric NAFLD.

In some embodiments of any of the compositions or methods disclosedherein, e.g., of any of the methods described herein, the subject has ahigh BMI, obesity, gut leakiness, gut dysbiosis, or gut microbiomedisturbance.

In some embodiments of any of the compositions or methods disclosedherein, e.g., of any of the methods described herein, the subject hascirrhosis, hepatocarcinoma, an increased risk of liver failure, anincreased risk of death, metabolic syndrome, or type 2 diabetes.

In some embodiments of any of the compositions or methods disclosedherein, e.g., of any of the methods described herein, the subject hasincreased levels of inflammatory cytokines relative to a normal subject,e.g., the subject has increased levels of TNFα relative to a normalsubject e.g., without the one or more symptoms or without the fattyliver disease.

In some embodiments, e.g., of any of the methods described herein, thesubject exhibits muscle atrophy or has a decreased ratio of muscletissue to adipose tissue relative to a normal subject, e.g., without theone or more symptoms or without a fatty liver disease, e.g., the subjectexhibits muscle atrophy without one or both of fibrosis or cirrhosis.

In some embodiments, e.g., of any of the methods described herein, thesubject exhibits reverse lipid transport from adipose tissue to livertissue.

In some embodiments, e.g., of any of the methods described herein, thesubject is treated with a composition, e.g., any composition asdescribed herein. In some embodiments of any of the aspects describedherein:

-   -   (i) an amino acid entity (e.g., at least one, two, or three of        the amino acid entities) of (a) is selected from Table 2; and/or    -   (ii) one or both of the R-amino acid entity and the Q-amino acid        entity are present at a higher amount (wt. %) than the L-amino        acid entity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B are graphs showing the effect of treatment with an aminoacid composition (Amino Acid Composition A-1) on the NAFLD activityscore, ballooning, and fibrosis in the STAM mouse model (FIG. 1A) and inthe FATZO mouse model (FIG. 1B).

FIG. 2 is a schematic showing the metabolic symptoms of patients withnon-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis(NASH), and cirrhosis prior to administration of a compositioncomprising amino acid entities described herein (top) and theimprovement in patients with NAFLD, NASH, and cirrhosis afteradministration of the composition (bottom).

FIG. 3 is a schematic showing treatment regimens for administration ofan amino acid composition to STAM and FATZO mice.

FIGS. 4A-4E are a series of graphs and images showing the effect oftreating STAM and FATZO mice with an amino acid composition on the NAFLDactivity score (NAS), steatosis, inflammation, and liver fibrosis asdetermined with histology.

FIGS. 5A-5B are images showing the levels of liver unsaturated fattyacids and acylcarnitines of STAM mice treated with the amino acidcomposition.

FIG. 6 is an image of a gene map of the liver gene expression patternfollowing treatment with the amino acid composition in STAM mice showingactivation of ACOX1.

FIGS. 7A-7D are images of gene maps of the liver gene expression patternfollowing treatment with the amino acid composition in STAM mice showingupstream regulator activation of anti-inflammatory IL-10 (FIG. 7A);inhibition of pro-inflammatory NF-kB (FIG. 7B), interferons, IL-1b, andIL-2 (FIG. 7C); and suppression of the fibrogenic TGF-b signalingpathway.

FIG. 8 is a series of graphs showing MCP-1 and MIP-1 protein levels,which are the ligands of C—C chemokine receptor types 2 (CCR2) and 5(CCR5), following treatment with the amino acid composition.

FIGS. 9A-9L are a series of microscopy images shown lipid accumulationin primary human hepatocytes following treatment with vehicle control(FIGS. 9A-9D), a LIVRQNAC amino acid composition (FIGS. 9E-9H), or freefatty acids and TNFα (FF+TNF; FIGS. 9I-9L).

FIG. 10 is a series of microscopy images showing liver histology (H&Estain or Sirius Red stain for collagen deposition) from FATZO mice afteradministration of the indicated amino acid compositions.

FIG. 11 is a series of microscopy images showing liver histology fromFATZO mice after administration of the indicated amino acidcompositions.

FIG. 12 is a series of graphs showing NAFLD activity scores (top leftpanel), Sirius Red staining (top right panel), steatosis levels (bottomleft panel), inflammation levels (bottom middle panel), and ballooning(bottom right panel) observed in fixed liver tissues from FATZO miceafter administration of the indicated amino acid compositions.

DETAILED DESCRIPTION

The present invention provides, at least in part, methods andcompositions comprising at least four different amino acid entities. Insome embodiments, the composition is capable of one, two, three, four,five, or six or all of:

a) decreasing or preventing liver fibrosis;

b) decreasing or preventing liver injury;

c) decreasing or preventing hepatocyte inflammation;

d) improving, e.g., increasing, glucose tolerance;

e) decreasing or preventing steatosis;

f) decreasing or preventing hepatocyte ballooning; or

g) improving gut function.

In some embodiments, at least one amino acid entity in the compositionsis not a peptide of more than 20 amino acid residues in length.

In some embodiments, the composition comprises a leucine (L)-amino acidentity, an arginine (R)-amino acid entity, a glutamine (Q)-amino acidentity; and an antioxidant or reactive oxygen species (ROS) scavenger(e.g., a N-acetylcysteine (NAC) entity, e.g., NAC). In some embodiments,at least one amino acid entity is not a peptide of more than 20 aminoacid residues in length. In some embodiments, the composition is capableof improving gut barrier function.

The composition described herein can be administered to a subject toprovide a beneficial effect in one or both of improving liver functionor treating (e.g., revering, reducing, ameliorating, or preventing) aliver disease (e.g., a fatty liver disease). A subject that may betreated with the composition include a subject having non-alcoholicfatty liver disease (NAFLD; e.g., pediatric NAFLD), such as a subjectwith non-alcoholic steatohepatitis (NASH) or NAFL, or subjects withalcoholic fatty liver disease (AFLD), such as alcoholic steatohepatitis(ASH). In particular, the subject may have one, two, or more (e.g., all)of a high BMI, obesity, fibrosis, or cirrhosis. The subject may alsohave one, two, or more (e.g., all) of gut leakiness, gut dysbiosis, orgut microbiome disturbance.

The subject may exhibit an improvement in liver function or liverdisease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) afteradministration of a composition comprising a L-amino acid entity, aR-amino acid entity, a Q-amino acid entity; and an antioxidant or ROSscavenger, e.g., a NAC entity, e.g., NAC. For example, the amino acidentity composition may be administered to the subject for a treatmentperiod of, e.g., two weeks, three weeks, four weeks, five weeks, sixweeks, seven weeks, eight weeks, nine weeks, 10 weeks, 11 weeks, 12weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, or longer at a dose ofabout 15 total grams per day to about 90 total grams per day (e.g., atotal of about 48 g or a total of about 72 g per day).

Treatment with the amino acid entity composition can result in improvedliver function in a subject, e.g., by one, two, three, four, five ormore (e.g., all) of increasing free fatty acid and lipid metabolism,improving mitochondrial function, browning of white adipose tissue(WAT), decreasing reactive oxygen species (ROS), increasing levels ofglutathione (GSH), decreasing hepatic inflammation, improving gutbarrier function, increasing insulin secretion, or improving glucosetolerance.

In some embodiments, the composition is for use as a medicament inimproving liver function in a subject (e.g., a subject with a liverdisease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)). In someembodiments, the composition including amino acid entities is for use asa medicament in treating (e.g., reversing, reducing, ameliorating, orpreventing) a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD(e.g., ASH)) in a subject.

In some embodiments, the composition is for use in the manufacture of amedicament for improving liver function in a subject (e.g., a subjectwith a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g.,ASH)). In some embodiments, the composition including amino acidentities is for use in the manufacture of a medicament for treating(e.g., reversing, reducing, ameliorating, or preventing) a liver disease(e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)) in a subject.

Additionally, the compositions can be used in methods of dietarymanagement of a subject (e.g., a subject without a liver disease or witha liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)).

One embodiment provides a nutritional supplement, dietary formulation,functional food, medical food, food, or beverage comprising acomposition described herein. Another embodiment provides a nutritionalsupplement, dietary formulation, functional food, medical food, food, orbeverage comprising a composition described herein for use in themanagement of any of the diseases or disorders described herein.

One embodiment provides a method of maintaining or improving liverhealth comprising administering to a subject an effective amount of acomposition described herein. Another embodiment provides a method ofproviding nutritional support or supplementation to a subject with aliver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH))comprising administering to the subject an effective amount of acomposition described herein. Yet another embodiment provides a methodof providing nutritional supplementation that aids in the management ofliver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH))comprising administering to the subject in need thereof an effectiveamount of a composition described herein.

Definitions

Terms used in the claims and specification are defined as set forthbelow unless otherwise specified.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an” and “the” include plural referentsunless the context clearly dictates otherwise.

As used herein, the term “amino acid entity” refers to an amino acid inone or both of free form or salt form, an amino acid residue of apeptide (e.g., of a dipeptide, oligopeptide, or polypeptide), aderivative of an amino acid, a precursor of an amino acid, or ametabolite of an amino acid.

As used herein the term “XXX amino acid entity” refers to an amino acidentity that if a free amino acid, comprises free XXX or XXX in saltform; if a peptide, refers to a peptide comprising an XXX residue; if aderivative, refers to a derivative of XXX; if a precursor, refers to aprecursor of XXX; and if a metabolite, refers to a XXX metabolite. Forexample, where XXX is leucine (L), then L-amino acid entity refers tofree L or L in salt form, a peptide comprising a L residue, a Lderivative, a L precursor, or a metabolite of L; where XXX is arginine(R), then R-amino acid entity refers to free R or R in salt form, apeptide comprising a R residue, a R derivative, a R precursor, or ametabolite of R; where XXX is glutamine (Q), then Q-amino acid entityrefers to free Q or Q in salt form, a peptide comprising a Q residue, aQ derivative, a Q precursor, or a metabolite of Q; and where XXX isN-acetylcysteine (NAC), then NAC-amino acid entity refers to free NAC orNAC in salt form, a peptide comprising a NAC residue, a NAC derivative,a NAC precursor, or a metabolite of NAC.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Exemplary degrees of error are within 20 percent (%),typically, within 10%, and more typically, within 5% of a given value orrange of values.

An “amino acid” refers to an organic compound having an amino group(—NH₂), a carboxylic acid group (—C(═O)OH), and a side chain bondedthrough a central carbon atom, and includes essential and non-essentialamino acids, as well as natural and unnatural amino acids.

The proteogenic amino acids, shown below, are known by three- andone-letter abbreviations in addition to their full names. For a givenamino acid, these abbreviations are used interchangeably herein. Forexample, Leu, L or leucine all refer to the amino acid leucine; Ile, Ior isoleucine all refer to the amino acid isoleucine; Val, V or valineall refer to the amino acid valine; Arg, R or arginine all refer to theamino acid arginine; and Gln, Q or glutamine all refer to the amino acidglutamine.

Likewise, the non-natural amino acid derivative N-acetylcysteine may bereferred to interchangeably by “NAC” or “N-acetylcysteine.”

Amino acids may be present as D- or L-isomers. Unless otherwiseindicated, amino acids referred to herein are L-isomers of amino acids.

TABLE 1 Amino acid names and abbreviations. Amino acid Three-letterOne-letter Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acidAsp D Cysteine Cys C Glutamic acid Glu E Glutamine Gln Q Glycine Gly GHistidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K MethionineMet M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr TTryptophan Trp W Tyrosine Tyr Y Valine Val V

The term “effective amount” as used herein means an amount of an aminoacid, or pharmaceutical composition which is sufficient enough tosignificantly and positively modify the symptoms and/or conditions to betreated (e.g., provide a positive clinical response). The effectiveamount of an active ingredient for use in a pharmaceutical compositionwill vary with the particular condition being treated, the severity ofthe condition, the duration of treatment, the nature of concurrenttherapy, the particular active ingredient(s) being employed, theparticular pharmaceutically-acceptable excipient(s) and/or carrier(s)utilized, and like factors with the knowledge and expertise of theattending physician.

A “pharmaceutical composition” described herein comprises at least oneamino acid and a pharmaceutically acceptable carrier or excipient. Insome embodiments, the pharmaceutical composition is used as atherapeutic, a nutraceutical, a medical food, or as a supplement.

The term “pharmaceutically acceptable” as used herein, refers to aminoacids, materials, excipients, compositions and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

A composition, formulation or product is “therapeutic” if it provides abeneficial clinical effect. A beneficial clinical effect can be shown bylessening the progression of a disease and/or alleviating one or moresymptoms of the disease.

A “unit dose” or “unit dosage” as used herein means an amount or dose ofmedicine prepared in an individual packet or container for convenience,safety, or monitoring. A “unit dose” or “unit dosage” comprises the drugproduct or drug products in the form in which they are marketed for use,with a specific mixture of active ingredients and inactive components(excipients), in a particular configuration (such as a capsule shell,for example), and apportioned into a particular dose.

As used herein, the terms “treat,” “treating,” or “treatment” of a liverdisease refer in one embodiment, to ameliorating, e.g., NAFLD (e.g.,NASH or NAFL) or AFLD (e.g., ASH), (i.e., slowing or arresting orreducing the development of the disease or at least one of the clinicalsymptoms thereof). In another embodiment, “treat,” “treating,” or“treatment” refers to alleviating or ameliorating at least one physicalparameter including those which may not be discernible by the patient.In yet another embodiment, “treat,” “treating,” or “treatment” refers tomodulating a symptom of a liver disease (e.g., NAFLD (e.g., NASH orNAFL) or AFLD (e.g., ASH)), either physically, (e.g., stabilization of adiscernible symptom), physiologically, (e.g., stabilization of aphysical parameter), or both. In yet another embodiment, “treat,”“treating,” or “treatment” refers to preventing or delaying the onset ordevelopment or progression of a liver disease (e.g., NAFLD (e.g., NASHor NAFL) or AFLD (e.g., ASH)).

Determination of Amino Acid Weight Percent and Amino Acid Ratios in aComposition

The weight ratio of a particular amino acid or particular amino acids ina composition or mixture of amino acids is the ratio of the weight ofthe particular amino acid or amino acids in the composition or mixturecompared to the total weight of amino acids present in the compositionor mixture. This value is calculated by dividing the weight of theparticular amino acid or of the particular amino acids in thecomposition or mixture by the weight of all amino acids present in thecomposition or mixture.

Compositions comprising Amino Acid Entities

The present disclosure provides compositions, e.g., pharmaceuticalcompositions, comprising amino acid entities. These pharmaceuticalcompositions are made up of amino acid entities including amino acids inone or both of free form or salt form, amino acid residues of a peptide(e.g., of a dipeptide, oligopeptide, or polypeptide), derivatives of anamino acid, precursors of an amino acid, or metabolites of an aminoacid. For example, the compositions can include a leucine (L)-amino acidentity, a arginine (R)-amino acid entity, a glutamine (Q)-amino acidentity; and an antioxidant or reactive oxygen species (ROS) scavenger,e.g., a N-acetylcysteine (NAC) entity, e.g., NAC (Table 2). Inparticular, at least one amino acid entity is not a peptide of more than20 amino acid residues in length.

TABLE 2 Amino acid entities include amino acids, precursors,metabolites, and derivatives of the compositions described herein.Exemplary Amino Acid Precursors Metabolites Derivatives L L-LeucineOxo-leucine HMB (beta- D-Leucine; N- hydroxy-beta- Acetyl-Leucinemethybutyrate); Oxo-leucine; Isovaleryl-CoA I L-Isoleucine2-Oxo-3-methyl- 2-Oxo-3-methyl- D-Isoleucine; N- valerate; Threoninevalerate; Acetyl- Methylbutyrl-CoA Isoleucine V L-Valine 2-Oxo-valerateIsobutryl-CoA; 3- D-Valine; N- HIB-CoA; 3-HIB Acetyl-Valine R L-ArginineArgininosuccinate; Ornithine; D-Arginine; N- Citrulline; Aspartate;Citrulline; Acetyl-Arginine; Glutamate Agmatine; Creatine Q L-GlutamineGlutamate Carbamoyl-P; D-Glutamine; N- Glutamate Acetyl- Glutamine; NACN- Serine; Acetylserine; Glutathione; D-Cysteine; L- AcetylcysteineCystathionine; Cystathionine; Cysteine; Homocysteine; Cystine;Methionine Cysteamine S L-Serine Phosphoserine, P- Glycine,hydroxypyruvate, L- Tryptophan, Glycine Acetylserine, Cystathionine,Phosphatidylserine

It is contemplated that alternatives to serine that can be an S-aminoacid entity include, for example, glycine, threonine, or a combinationof serine and glycine (e.g., a 1:1 ratio of serine and glycine).

In some embodiments, the total weight of the L-amino acid entity,R-amino acid entity, Q-amino acid entity; and ROS scavenger, e.g., a NACentity, e.g., NAC, is greater than the total wt. of other amino acidentities in the composition. In certain embodiments, two, three, or more(e.g., all) of methionine (M), trytophan (W), or valine (V) may beabsent from the amino acid entity composition, or if present, arepresent at less than 2 weight (wt.) %.

In some embodiments, one or both of the R-amino acid entity and theQ-amino acid entity are present at a higher amount (wt. %) than theL-amino acid entity. The R-amino acid entity can be present, e.g., at anamount of at least 2 wt. %, at least 3 wt. %, at least 4 wt. %, at least5 wt. %, at least 6 wt. %, at least 7 wt. %, or at least 8 wt. % greaterthan the L-amino acid entity. The Q-amino acid entity can be present,e.g., at an amount of at least 2 wt. %, at least 3 wt. %, at least 4 wt.%, or at least 5 wt. % greater than the L-amino acid entity.

In some embodiments, the L-amino acid entity is selected from the groupconsisting of a precursor, a metabolite, and a derivative. In certainembodiments, the L-amino acid entity is selected from the groupconsisting of L-leucine, β-hydroxy-β-methybutyrate (HMB), oxo-leucine,isovaleryl-CoA, D-leucine, and n-acetylleucine. In one embodiment, theL-amino acid entity is L-leucine. In another embodiment, the L-aminoacid entity is HMB.

In some embodiments, the R-amino acid entity is selected from the groupconsisting of a precursor, a metabolite, and a derivative. In certainembodiments, the R-amino acid entity is selected from the groupconsisting of L-arginine, D-arginine, ornithine, argininosuccinate,citrulline, aspartate, glutamate, agmatine, and N-acetyl-arginine. Inone embodiment, the R-amino acid entity is L-arginine. In oneembodiment, the R-amino acid entity is creatine. In another embodiment,the R-amino acid entity is ornithine.

In some embodiments, the Q-amino acid entity is selected from the groupconsisting of a precursor, a metabolite, and a derivative. In certainembodiments, the Q-amino acid entity is selected from the groupconsisting of L-glutamine, glutamate, carbamoyl-P, glutamate,D-glutamine, and n-acetylglutamine. In one embodiment, the Q-amino acidentity is L-glutamine.

In some embodiments, the NAC-amino acid entity is selected from thegroup consisting of a precursor, a metabolite, and a derivative. Incertain embodiments, the NAC-amino acid entity is selected from thegroup consisting NAC, serine, acetylserine, cystathionine,cystathionine, homocysteine, methionine, glutathione, D-cysteine, andL-cysteine. In one embodiment, the NAC entity is NAC. In one embodiment,the NAC entity is glutathione.

In various embodiments, the composition further comprises one or twoadditional branched-chain amino acid (BCAA)-entities, e.g., one or bothof an isoleucine (I)-amino acid-entity and a valine (V)-aminoacid-entity. In some embodiments, both the I-amino acid-entity and theV-amino acid-entity are present. In certain embodiments, the L-entity ispresent at a higher amount (% by weight) than one or both of the I-aminoacid-entity and the V-amino acid-entity (e.g., the L-entity is presentat an amount of at least 10 wt. %, at least 15 wt. %, at least 20 wt. %,at least 25 wt. %, at least 30 wt. %, at least 35 wt. %, at least 40 wt.%, at least 45 wt. %, or at least 50 wt. % greater than one or both ofthe I-amino acid-entity and the V-amino acid-entity).

In some embodiments, the I-amino acid entity is selected from the groupconsisting of a salt, a precursor, a metabolite, and a derivative. Incertain embodiments, the I-amino acid entity is selected from the groupconsisting of L-isoleucine, 2-oxo-3-methyl-valerate, threonine,2-oxo-3-methyl-valerate, methylbutyrl-CoA, D-isoleucine, andN-acetyl-isoleucine. In one embodiment, the I-amino acid entity isL-isoleucine.

In some embodiments, the V-amino acid entity is selected from the groupconsisting of a precursor, a metabolite, and a derivative. In certainembodiments, the V-amino acid entity is selected from the groupconsisting of L-valine, 2-oxo-valerate, isobutryl-CoA, 3-HIB-CoA, 3-HIB,D-valine, and N-acetyl-valine. In one embodiment, the I-amino acidentity is L-valine.

In some embodiments, the composition comprises L-leucine or a leucinemetabolite (e.g., HMB), L-arginine or an L-arginine metabolite (e.g.,creatine or ornithine), L-glutamine, and NAC or a NAC metabolite, e.g.,glutathione. In one embodiment, the composition comprises L-leucine,L-arginine, L-glutamine, and NAC. In one embodiment, the compositioncomprises HMB, creatine, L-glutamine, and glutathione. In oneembodiment, the composition comprises HMB, ornithine, L-glutamine, andglutathione. In one embodiment, the composition comprises HMB,L-arginine, L-glutamine, and NAC. In one embodiment, the compositioncomprises L-leucine, creatine, L-glutamine, and NAC. In one embodiment,the composition comprises L-leucine, ornithine, L-glutamine, and NAC. Inone embodiment, the composition comprises L-leucine, L-arginine,L-glutamine, and glutathione.

In some embodiments, the weight (wt.) ratio of the L-amino acid entity,the R-amino acid entity, the Q-amino acid entity, and the NAC-amino acidentity is about 0.5 to 3:0.5 to 4:1 to 4:0.1 to 2.5. In one embodiment,the wt. ratio of the L-amino acid entity, the R-amino acid entity, theQ-amino acid entity, and the NAC-amino acid entity is about1:1.5:2:0.15.

In some embodiments, the wt. ratio of the L-amino acid entity, theI-amino acid entity, the V-amino acid entity, the R-amino acid entity,the Q-amino acid entity, and the NAC-amino acid entity is about 0.5 to2:0.1 to 1:0.1 to 1:0.5 to 3:0.5 to 4:0.1 to 0.5. In an embodiment, thewt. ratio of the L-amino acid entity, the I-amino acid entity, theV-amino acid entity, the R-amino acid entity, the Q-amino acid entity,and the NAC-amino acid entity is about 1:0.5:0.5:1.5:2:0.15.

In various embodiments, the total wt. of amino acids present is about 2g to about 60 g. In certain embodiments, the total wt. of amino acidspresent is about 6 g, about 12 g, about 18 g, about 24 g, or about 48 g.In one embodiment, the total wt. of amino acids present is about 6 g. Inone embodiment, the total wt. of amino acids present is about 12 g. Inone embodiment, the total wt. of amino acids present is about 18 g. Inan embodiment, the total wt. of amino acids present is about 24 g. Inone embodiment, the total wt. of amino acids present is about 48 g.

In some embodiments, the composition comprises about 0.5 g to about 10 gof the L-amino acid entity, about 0.25 g to about 5 g of the I-aminoacid entity, about 0.25 g to about 5 g of the V-amino acid entity, about1 g to about 20 g of the R-amino acid entity, about 1 g to about 20 g ofthe Q-amino acid entity, and about 0.1 g to about 5 g of the NAC-aminoacid entity. In an embodiment, the composition comprises about 1 g ofthe L-amino acid entity, about 0.5 g of the I-amino acid entity, about0.5 g of V-amino acid entity, about 1.5 g of R-amino acid entity, about2 g of Q-amino acid entity, and about 0.15 g of NAC-amino acid entity.In an embodiment, the composition comprises about 2 g of the L-aminoacid entity, about 1 g of the I-amino acid entity, about 1 g of theV-amino acid entity, about 3 g of the R-amino acid entity, about 4 g ofthe Q-amino acid entity, and about 0.3 g of the NAC-amino acid entity.In an embodiment, the composition comprises about 4 g of the L-aminoacid entity, about 2 g of the I-amino acid entity, about 2 g of theV-amino acid entity, about 6 g of the R-amino acid entity, about 8 g ofthe Q-amino acid entity, and about 0.6 g of the NAC-amino acid entity.

In some embodiments, the amino acids comprise about 10 wt % to about 30wt % leucine, about 5 wt % to about 15 wt % isoleucine, about 5 wt % toabout 15 wt % valine, about 15 wt % to about 40 wt % arginine, about 20wt % to about 50 wt % glutamine, and about 1 wt % to about 8 wt %n-acetylcysteine. In certain embodiments, the amino acids comprise about16 wt % to about 18 wt % leucine, about 7 wt % to about 9 wt %isoleucine, about 7 wt % to about 9 wt % valine, about 28 wt % to about32 wt % arginine, about 31 wt % to about 34 wt % glutamine, and about 1wt % to about 5 wt % n-acetylcysteine. In an embodiment, the amino acidscomprise about 16.8 wt % leucine, about 8.4 wt % isoleucine, about 8.4wt % valine, about 30.4 wt % arginine, about 33.6 wt % glutamine, andabout 2.5 wt % N-acetylcysteine.

In any of the foregoing embodiments, at least one amino acid entity is afree amino acid, e.g., one, two, three, or more (e.g., all) amino acidentities are a free amino acid. In some embodiments, the L-amino acidentity, the R-amino acid entity, the Q-amino acid entity, and theNAC-amino acid entity is a free amino acid entity. In certainembodiment, the L-amino acid entity, the I-amino acid entity, theV-amino acid entity, the R-amino acid entity, the Q-amino acid entity,and the NAC-amino acid entity a free amino acid.

In any of the foregoing embodiments, at least one amino acid entity isin a salt form, e.g., one, two, three, or more (e.g., all) of the aminoacid entities is in a salt form. In some embodiments, wherein theL-amino acid entity, the R-amino acid entity, the Q-amino acid entity,and the NAC-amino acid entity is in a salt form. In certain embodiments,the L-amino acid entity, the I-amino acid entity, the V-amino acidentity, the R-amino acid entity, the Q-amino acid entity, and theNAC-amino acid entity is in a salt form.

In any of the foregoing embodiments, the composition comprises acombination of 2 to 20 different amino acid entities, e.g., 5 to 15different amino acid entities.

In some embodiments, the NAC entity is more stable than cysteine. Incertain embodiments, the NAC entity does not comprise cysteine.

In some embodiments, the composition further comprises one, two, three,four, five, six, seven, eight, nine, ten, or more (e.g., all) or more ofserine, glycine, glutamine, HMB, arginine, L-leucine, citrulline,glutamine, ornithine, L-cysteine, cystine, or glutathione.

In some embodiments, the composition further comprises serine.

In some embodiments, the composition further comprises glycine.

In some embodiments, the composition further comprises carnitine.

In some embodiments, the composition includes arginine, glutamine,N-acetylcysteine, and a branched-chain amino acid (BCAA) chosen fromone, two, or all of leucine, isoleucine, and valine.

In some embodiments, the BCAA is leucine.

In some embodiments, the BCAA is isoleucine.

In some embodiments, the BCAA is valine.

In some embodiments, the BCAA is leucine and isoleucine.

In some embodiments, the BCAA is leucine and valine.

In some embodiments, the BCAA is isoleucine and valine.

In some embodiments, the BCAA is leucine, isoleucine, and valine.

In particular, the composition may consist of leucine, isoleucine,valine, arginine, glutamine, and N-acetylcysteine.

In some embodiments, the amino acids leucine, isoleucine, valine,arginine, glutamine and N-acetylcysteine are present in a weight ratioof about 1:0.5:0.5:1.5:2:0.1-0.3. In some embodiments, the amino acidsleucine, isoleucine, valine, arginine, glutamine and N-acetylcysteineare present in a weight ratio of about 1:0.5:0.5:1.5:2:0.15. In someembodiments, the amino acids leucine, isoleucine, valine, arginine,glutamine and N-acetylcysteine are present in a weight ratio of about1:0.5:0.5:1.5:2:0.25.

In some embodiments, the amino acids leucine, isoleucine, valine,arginine (e.g., arginine HCl), glutamine and N-acetylcysteine arepresent in a weight ratio of about 1:0.5:0.5:1.5-1.81:2:0.1-0.3. In someembodiments, the amino acids leucine, isoleucine, valine, arginine(e.g., arginine HCl), glutamine and N-acetylcysteine are present in aweight ratio of about 1:0.5:0.5:1.5-1.81:2:0.15. In some embodiments,the amino acids leucine, isoleucine, valine, arginine (e.g., arginineHCl), glutamine, and N-acetylcysteine are present in a weight ratio ofabout 1:0.5:0.5:1.5-1.81:2:0.25.

In some embodiments, the amino acids leucine, isoleucine, valine,arginine (e.g., arginine HCl), glutamine and N-acetylcysteine arepresent in a weight ratio of about 1:0.5:0.5:1.81:2:0.1-0.3. In someembodiments, the amino acids leucine, isoleucine, valine, arginine(e.g., arginine HCl), glutamine and N-acetylcysteine are present in aweight ratio of about 1:0.5:0.5:1.81:2:0.15. In some embodiments, theamino acids leucine, isoleucine, valine, arginine (e.g., arginine HCl),glutamine, and N-acetylcysteine are present in a weight ratio of about1:0.5:0.5:1.81:2:0.25.

In some embodiments, the amino acids leucine, isoleucine, valine,arginine HCl, glutamine and N-acetylcysteine are present in a weightratio of about 1:0.5:0.5:1.5:2:0.1 to 0.3. In some embodiments, theamino acids leucine, isoleucine, valine, arginine HCl, glutamine andN-acetylcysteine are present in a weight ratio of about1:0.5:0.5:1.5:2:0.25. In some embodiments, the amino acids leucine,isoleucine, valine, arginine HCl, glutamine and N-acetylcysteine arepresent in a weight ratio of 1:0.5:0.5:1.5:2:0.15.

In some embodiments, the amino acids leucine, isoleucine, valine,arginine HCl, glutamine and N-acetylcysteine are present in a weightratio of about 1:0.5:0.5:1.81:2:0.1 to 0.3. In some embodiments, theamino acids leucine, isoleucine, valine, arginine HCl, glutamine andN-acetylcysteine are present in a weight ratio of about1:0.5:0.5:1.81:2:0.25. In some embodiments, the amino acids leucine,isoleucine, valine, arginine HCl, glutamine and N-acetylcysteine arepresent in a weight ratio of 1:0.5:0.5:1.81:2:0.15.

In some embodiments, the amino acids leucine, isoleucine, valine,arginine, glutamine and N-acetylcysteine are present in a weight ratioof about 1:0.5:0.5:1.81:2:0.1-0.3. In some embodiments, the amino acidsleucine, isoleucine, valine, arginine, glutamine and N-acetylcysteineare present in a weight ratio of about 1:0.5:0.5:1.81:2:0.15. In someembodiments, the amino acids leucine, isoleucine, valine, arginine,glutamine, and N-acetylcysteine are present in a weight ratio of about1:0.5:0.5:1.81:2:0.25.

In some embodiments, the amino acids leucine, isoleucine, valine,arginine, glutamine and N-acetylcysteine are present in a weight ratioof about 1:0.5:0.5:1.5:2:0.1 to 0.3. In some embodiments, the aminoacids leucine, isoleucine, valine, arginine, glutamine andN-acetylcysteine are present in a weight ratio of about1:0.5:0.5:1.5:2:0.25. In some embodiments, the amino acids leucine,isoleucine, valine, arginine, glutamine and N-acetylcysteine are presentin a weight ratio of 1:0.5:0.5:1.5:2:0.15.

In some embodiments, a total weight (wt) of the amino acids is about 2 gto about 60 g.

In some embodiments, the total weight of amino acids present is about 5g, about 6 g, about 7 g, about 11 g, about 12 g, about 13 g, about 14 g,about 15 g, about 16 g, about 17 g, about 18 g, about 19 g, about 20 g,about 21 g, about 22 g, about 23 g, about 24 g, about 25 g, about 26 g,about 27 g, about 28 g, about 29 g, about 30 g, about 31 g, about 32 g,about 33 g, about 34 g, about 35 g, about 36 g, about 37 g, about 38 g,about 39 g, about 40 g, about 41 g, about 42 g, about 43 g, about 44 g,about 45 g, about 46 g, about 47 g, about 48 g, about 49 g, or about 50g.

In certain embodiments, the total wt of the amino acids is about 6 g.

In certain embodiments, the total wt of the amino acids is about 12 g.

In certain embodiments, the total wt of the amino acids is about 18 g.

In certain embodiments, the total wt of the amino acids is about 24 g.

In certain embodiments, the total wt of the amino acids is about 48 g.

In some embodiments, the composition includes about 0.5 g to about 10 gof leucine, about 0.25 g to about 5 g of isoleucine, about 0.25 g toabout 5 g of valine, about 1 g to about 20 g of arginine, about 1 g toabout 20 g of glutamine, and about 0.1 g to about 5 g ofN-acetylcysteine.

In some embodiments, the composition includes at least 1 g of leucine,at least 0.5 g of isoleucine, at least 0.5 g of valine, at least 1.5 gof arginine (or 1.81 g of arginine HCl), at least 2 g of glutamine, andat least 0.15 g of N-acetylcysteine.

In some embodiments, the composition includes about 1 g of leucine,about 0.5 g of isoleucine, about 0.5 g of valine, about 1.5 g ofarginine (or 1.81 g of arginine HCl), about 2 g of glutamine, and about0.15 g of N-acetylcysteine.

In some embodiments, the composition includes at least 2 g of leucine,at least 1 g of isoleucine, at least 1 g of valine, at least 3.0 g ofarginine (or 3.62 g of arginine HCl), at least 4 g of glutamine, and atleast 0.3 g of N-acetylcysteine.

In some embodiments, the composition includes about 2 g of leucine,about 1 g of isoleucine, about 1 g of valine, about 3.0 g of arginine(or 3.62 g of arginine HCl), about 4 g of glutamine, and about 0.3 g ofN-acetylcysteine.

In some embodiments, the composition includes at least 4 g of leucine,at least 2 g of isoleucine, at least 2 g of valine, at least 6.0 g orarginine (or 7.24 g of arginine HCl), at least 8 g of glutamine, and atleast 0.6 g of N-acetylcysteine.

In some embodiments, the composition includes about 4 g of leucine,about 2 g of isoleucine, about 2 g of valine, about 6.0 g or arginine(or 7.24 g of arginine HCl), about 8 g of glutamine, and about 0.6 g ofN-acetylcysteine.

In some embodiments, the composition includes at least 1.0 g of leucine,at least 0.5 g of isoleucine, at least 0.5 g of valine, at least 1.5 gof arginine, at least 2.0 g of glutamine, or at least 0.15 g ofN-acetylcysteine. In some embodiments, the composition includes about1.0 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine,about 1.5 g of arginine, about 2.0 g of glutamine, or about 0.15 g ofN-acetylcysteine.

In some embodiments, the composition includes at least 1.0 g of leucine,at least 0.5 g of isoleucine, at least 0.5 g of valine, at least 1.5 gof arginine, at least 2.0 g of glutamine, and at least 0.25 g ofN-acetylcysteine. In some embodiments, the composition includes about1.0 g of leucine, about 0.5 g of isoleucine, about 0.5 g of valine,about 1.5 g of arginine, about 2.0 g of glutamine, and about 0.25 g ofN-acetylcysteine.

In some embodiments, the amino acids of the composition include about 10wt % to about 30 wt % leucine, about 5 wt % to about 15 wt % isoleucine,about 5 wt % to about 15 wt % valine, about 15 wt % to about 40 wt %arginine, about 20 wt % to about 50 wt % glutamine, and about 1 wt % toabout 8 wt % N-acetylcysteine.

In some embodiments, the amino acids of the composition include about 16wt % to about 18 wt % leucine, about 7 wt % to about 9 wt % isoleucine,about 7 wt % to about 9 wt % valine, about 28 wt % to about 32 wt %arginine, about 31 wt % to about 34 wt % glutamine, and about 1 wt % toabout 5 wt % N-acetylcysteine.

In some embodiments, the amino acids of the composition include about16.8 wt % leucine, about 8.4 wt % isoleucine, about 8.4 wt % valine,about 30.4 wt % arginine, about 33.6 wt % glutamine, and about 2.5 wt %N-acetylcysteine.

In some embodiments, the composition comprises one or more excipientsselected from the group consisting of: citric acid, lecithin, asweetener, a dispersion enhancer, a flavoring, a bitterness maskingagent, and a natural or artificial coloring.

In some embodiments, the composition comprises citric acid.

In some embodiments, the composition is in the form of a solid, powder,solution, or gel. In certain embodiments, the composition is in the formof a powder (e.g. in a packet)

In some embodiments, the composition includes one or morepharmaceutically acceptable excipients, wherein the amino acids compriseleucine, arginine, glutamine, and N-acetylcysteine. An aspect of thepresent disclosure provides a composition comprising free amino acidsand one or more pharmaceutically acceptable excipients, wherein theamino acids consist of leucine, arginine, glutamine, andN-acetylcysteine. In some embodiments, the amino acids leucine,arginine, glutamine, N-acetylcysteine and glycine are present in aweight ratio of 1:1.5:2:0.15. In some embodiments, the compositioncomprises at least 1.0 g of leucine, at least 1.5 g of arginine, atleast 2.0 g of glutamine, or at least 0.15 g of N-acetylcysteine. Insome embodiments, the composition comprises at least 1.5 g of arginineand at least 2.0 g of glutamine. In some embodiments, the amino acidsleucine, arginine, glutamine, and N-acetylcysteine are present in weight% of each compared to total amino acid weight of 20.4 to 22.6%, 30.6 to33.9%, 40.9 to 45.2%, and 3.1 to 3.4%, respectively. In someembodiments, the amino acids leucine, arginine, glutamine, andN-acetylcysteine, are present in weight % of each compared to totalamino acid weight of 21.5%, 32.3%, 43.0%, and 3.2%, respectively.

In some embodiments, the composition further includes a farnesoid Xreceptor (FXR) agonist, a stearoyl CoA desaturase inhibitor, a CCR2 andCCR5 chemokine antagonist, a PPAR alpha and delta agonist, a caspaseinhibitor, a galectin-3 inhibitor, an acetyl CoA carboxylase inhibitor,or an ileal sodium bile acid co-transporter inhibitor. In someembodiments, the composition further comprises an FXR agonist. Incertain embodiments, the FXR agonist is obeticholic acid. In someembodiments, the composition further includes one or more of: LMB-763,LJN-452, emricasan, and cenicriviroc.

An exemplary Amino Acid Composition includes leucine, isoleucine,valine, arginine HCl, glutamine, and N-acetylcysteine as its amino acidentities in a wt. ratio of 1:0.5:0.5:1.81:2:0.15 (Table 3). An exemplaryAmino Acid Composition includes leucine, isoleucine, valine, arginine,glutamine, and N-acetylcysteine as its amino acid entities in a wt.ratio of 1:0.5:0.5:1.5:2:0.15 (Table 4).

TABLE 3 Exemplary amino acid components of the composition includingArginine HCl. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 16.78 1.00 g 2 g 4 g Isoleucine 0.5 8.39 0.50 g 1 g 2 g Valine0.5 8.39 0.50 g 1 g 2 g Arginine HCl 1.81 30.37 1.81 g 3.62 g   7.24 g  Glutamine 2 33.56 2.00 g 4 g 8 g N-acetylcysteine 0.15 2.52 0.15 g 0.3g   0.6 g   Total amino acids 5.96 g ~12 g    ~24 g   

TABLE 4 Exemplary amino acid components of the composition includingArginine. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 17.70 1.00 g 2 4 Isoleucine 0.5 8.85 0.50 g 1 2 Valine 0.58.85 0.50 g 1 2 Arginine 1.5 26.55  1.5 g 3 6 Glutamine 2 35.4 2.00 g 48 N-acetylcysteine 0.15 2.65 0.15 g 0.3   0.6   Total amino acids 5.65 g11.3 g  22.6 g 

An exemplary Amino Acid Composition includes leucine, isoleucine,valine, arginine HCl, glutamine, and N-acetylcysteine as its amino acidentities in a wt. ratio of 1:0.5:0.5:0.905:2:0.15 (Table 5). Anexemplary Amino Acid Composition includes leucine, isoleucine, valine,arginine, glutamine, and N-acetylcysteine as its amino acid entities ina wt. ratio of 1:0.5:0.5:0.75:2:0.15 (Table 6).

TABLE 5 Exemplary amino acid components of the composition includingArginine HCl. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 19.78 1.00 g 2 g 4 g Isoleucine 0.5 9.89 0.50 g 1 g 2 g Valine0.5 9.89 0.50 g 1 g 2 g Arginine HCl 0.905 17.90 0.905 g  1.81 g   3.62g   Glutamine 2 39.56 2.00 g 4 g 8 g N-acetylcysteine 0.15 2.97 0.15 g0.3 g   0.6 g   Total amino acids 5.06 g ~10 g    ~20 g   

TABLE 6 Exemplary amino acid components of the composition includingArginine. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 20.41 1.00 g 2 4 Isoleucine 0.5 10.20 0.50 g 1 2 Valine 0.510.20 0.50 g 1 2 Arginine 0.75 15.31 0.75 g 1.5   3 Glutamine 2 40.822.00 g 4 8 N-acetylcysteine 0.15 3.06 0.15 g 0.3   0.6   Total aminoacids  4.9 g 9.8 g 19.6 g 

An exemplary Amino Acid Composition includes leucine, isoleucine,valine, arginine HCl, glutamine, and N-acetylcysteine as its amino acidentities in a wt. ratio of 1:0.5:0.25:0.905:1:0.225 (Table 7). Anexemplary Amino Acid Composition includes leucine, isoleucine, valine,arginine, glutamine, and N-acetylcysteine as its amino acid entities ina wt. ratio of 1:0.5:0.25:0.75:1:0.225 (Table 8).

TABLE 7 Exemplary amino acid components of the composition includingArginine HCl. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 25.77 1.00 g   2 g 4 g Isoleucine 0.5 12.89 0.50 g   1 g 2 gValine 0.25 6.44 0.25 g 0.50 g 1 g Arginine HCl 0.905 23.32 0.905 g 1.81 g 3.62 g   Glutamine 1 25.77 1.00 g   2 g 4 g N-acetylcysteine0.225 5.80 0.225 g  0.45 g 0.9 g   Total amino acids 3.88 g 7.76 g 15.52g   

TABLE 8 Exemplary amino acid components of the composition includingArginine. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 26.85 1.00 g 2 4 Isoleucine 0.5 13.42 0.50 g 1 2 Valine 0.256.71 0.25 g 0.5   1 Arginine 0.75 20.13 0.75 g 1.5   3 Glutamine 1 26.851.00 g 2 4 N-acetylcysteine 0.225 6.04 0.225 g  0.45   0.9   Total aminoacids 3.725 g  7.45 g  14.9 g 

An exemplary Amino Acid Composition includes leucine, isoleucine,valine, arginine HCl, glutamine, N-acetylcysteine, and serine as itsamino acid entities in a wt. ratio of 1:0.5:0.25:0.905:1:0.225:0.667(Table 9). An exemplary Amino Acid Composition includes leucine,isoleucine, valine, arginine, glutamine, N-acetylcysteine, and serine asits amino acid entities in a wt. ratio of 1:0.5:0.25:0.75:1:0.225:1.5(Table 10).

TABLE 9 Exemplary amino acid components of the composition includingArginine HCl. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 18.59 1.00 g   2 g 4 g Isoleucine 0.5 9.29 0.50 g   1 g 2 gValine 0.25 4.65 0.25 g 0.50 g 1 g Arginine HCl 0.905 16.82 0.905 g 1.81 g 3.62 g   Glutamine 1 18.59 1.00 g   2 g 4 g N-acetylcysteine0.225 4.18 0.225 g  0.45 g 0.9 g   Serine 1.5 27.88 1.5   3 6   Totalamino acids 5.38 g 10.76 g  21.52 g   

TABLE 10 Exemplary amino acid components of the composition includingArginine. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 19.14 1.00 g 2 4 Isoleucine 0.5 9.57 0.50 g 1 2 Valine 0.254.78 0.25 g 0.5 1 Arginine 0.75 14.35 0.75 g 1.5 3 Glutamine 1 19.141.00 g 2 4 N-acetylcysteine 0.225 4.31 0.225 g  0.45 0.9 Serine 1.528.71 1.5  3 6 Total amino acids 5.225   10.45 20.9

An exemplary Amino Acid Composition includes leucine, isoleucine,valine, arginine HCl, glutamine, N-acetylcysteine, and serine as itsamino acid entities in a wt. ratio of 1:0.5:0.25:0.905:1:0.225:0.667(Table 11). An exemplary Amino Acid Composition includes leucine,isoleucine, valine, arginine, glutamine, N-acetylcysteine, and serine asits amino acid entities in a wt. ratio of 1:0.5:0.25:0.75:1:0.225:1.667(Table 12).

TABLE 11 Exemplary amino acid components of the composition includingArginine HCl. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 18.02 1.00 g   2 g 4 g Isoleucine 0.5 9.01 0.50 g   1 g 2 gValine 0.25 4.50 0.25 g 0.50 g 1 g Arginine HCl 0.905 16.31 0.905 g 1.81 g 3.62 g   Glutamine 1 18.02 1.00 g   2 g 4 g N-acetylcysteine0.225 4.05 0.225 g  0.45 g 0.9 g   Serine 1.667 30.09 1.67 g 3.33 g 6.67g   Total amino acids 5.55 g 11.09 g  22.19 g   

TABLE 12 Exemplary amino acid components of the composition includingArginine. Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2Leucine 1 18.54 1.00 g 2 4 Isoleucine 0.5 9.27 0.50 g 1 2 Valine 0.254.64 0.25 g 0.5   1 Arginine 0.75 13.91 0.75 g 1.5   3 Glutamine 1 18.541.00 g 2 4 N-acetyl- 0.225 4.17 0.225 g  0.45   0.9   cysteine Serine1.667 30.92 1.67 g 3.33 g  6.67 g  Total amino 5.395 g  10.78 g  21.57g  acids

The disclosure also provides a composition including at least fourdifferent amino acid entities (e.g., four, five, six, or more differentamino acid entitites), in which the composition is capable of one, two,three, four, five, or all of:

a) one or both of decreasing or preventing one or both of liver fibrosisor liver injury;

b) one or both of decreasing or preventing hepatocyte inflammation;

c) improving, e.g., increasing, glucose tolerance;

d) one or both of decreasing or preventing steatosis; or

e) one or both of decreasing or preventing hepatocyte ballooning,

provided that at least one amino acid entity is not a peptide of morethan 20 amino acid residues in length.

In some embodiments, the composition includes at least four differentamino acid entities (e.g., four, five, six, or more different amino acidentities) that decreases or prevents one or both of liver fibrosis orliver injury. For instance, the reducing and/or inhibiting liverfibrosis and/or liver injury comprises can include reducing a level ofone or both of collagen, e.g., type I and III collagen or α-smoothmuscle actin (αSMA).

In some embodiments, the composition includes at least four differentamino acid entities (e.g., four, five, six, or more different amino acidentities) that decreases or prevents hepatocyte inflammation. In someembodiments, the reducing and/or inhibiting liver fibrosis and/or liverinjury includes reducing a level or activity of one, two, three, four,or more (e.g., all) of a matrix metalloproteinase (MMP) (e.g., MMP-13,MMP-2, MMP-9, MT1-MMP, MMP-3, or MMP-10), a tissue inhibitor ofmetalloproteinase (TIMP) (e.g., TIMP1), aspartate transaminase (AST),alanine transaminase (ALT), or N-terminal fragment of type III collagen(proC3).

In some embodiments, the decreasing or preventing hepatocyteinflammation comprises reducing a level or activity of one, two, three,four, five, six, seven or more (e.g., all) of NF-kB, interferons, IL-1b,IL-2, MCP-1, MIP-1, a caspase-cleaved keratin 18 fragments (e.g., one orboth of M30 or M65), or C-reactive protein. In an embodiment, thedecreasing or preventing hepatocyte inflammation comprises increasing alevel or activity of IL-10.

In an embodiment, the improving, e.g., increasing, glucose tolerance,comprises increasing a level or activity of adiponectin. In anembodiment, the improving, e.g., increasing, glucose tolerance,comprises decreasing a level or activity of FGF-21.

In certain embodiments, the hepatocyte inflammation comprises LPSinduced hepatocyte inflammation.

In some embodiments, the composition is capable of enhancing fatty acidoxidation, e.g., one or both of reducing levels of unsaturated fattyacids or increasing levels of acylcarnitine (e.g., in a STAM mouse modelor a FATZO mouse model). In certain embodiments, the reduction in levelsof unsaturated fatty acids is at least 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the level ofchange shown in Table 53, e.g., measured as described in Example 9. Incertain embodiments, the increase in levels of acylcarnitine is at least25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%,95%, or 99% of the level of change shown in Table 53, e.g., measured asdescribed in Example 9.

In certain embodiments, the composition is capable of reducing, orreduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected usingan assay of alanine transaminase (ALT), e.g., an antibody-baseddetection assay, e.g., an ELISA, e.g., as described in Example 4, e.g.,relative to a reference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, orreduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected usingan assay of aspartate transaminase (AST), e.g., an antibody-baseddetection assay, e.g., an ELISA, e.g., as described in Example 4, e.g.,relative to a reference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, orreduces, alanine transaminase (ALT) by at least 5%, 10%, 15%, 20%, 21%,22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using anassay of ALT, e.g., an antibody-based detection assay, e.g., an ELISA,e.g., as described in Example 4, e.g., relative to a referencecomposition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, orreduces, aspartate transaminase (AST) by at least 5%, 10%, 15%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected usingan assay of AST, e.g., an antibody-based detection assay, e.g., anELISA, e.g., as described in Example 4, e.g., relative to a referencecomposition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, orreduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%,21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected usingan assay of hydroxyproline, e.g., an antibody-based detection assay,e.g., an ELISA, e.g., as described in Example 4, e.g., relative to areference composition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, orreduces, hydroxyproline levels by at least 5%, 10%, 15%, 20%, 21%, 22%,23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay ofhydroxyproline, e.g., an antibody-based detection assay, e.g., an ELISA,e.g., as described in Example 4, e.g., relative to a referencecomposition (e.g., a vehicle control).

In certain embodiments, the composition is capable of reducing, orreduces, liver fibrosis or liver injury by at least 5%, 10%, 15%, 20%,20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% asdetected using LX-2 cells, e.g., levels of Col1a1, Acta2, and/or TIMP2in LX-2 cells, e.g., as assessed using a nucleic acid amplificationmethod, e.g., PCR or qRT-PCR, e.g., as described in Example 7, e.g.,relative to a reference composition (e.g., a vehicle control; an aminoacid composition comprising L-leucine, L-isoleucine, L-valine; NAC; oran amino acid composition comprising L-arginine, L-glutamine, and NAC).

In certain embodiments, the composition is capable of reducing, orreduces, expression of one or more collagen biomarkers (e.g., Col1a1,Acta2, and/or TIMP2) by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using LX-2cells, e.g., levels of Col1a1, Acta2, and/or TIMP2 in LX-2 cells, e.g.,as assessed using a nucleic acid amplification method, e.g., PCR orqRT-PCR, e.g., as described in Example 7, e.g., relative to a referencecomposition (e.g., a vehicle control; an amino acid compositioncomprising L-leucine, L-isoleucine, L-valine; an amino acid compositioncomprising L-arginine, L-glutamine, and NAC; or NAC).

In certain embodiments, the composition is capable of reducing, orreduces, expression of one or more collagen biomarkers (e.g., Col1a1) byat least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, or 90% as detected using primary hepatic stellate cells,e.g., levels of Col1a1 in primary hepatic stellate cells, e.g., asassessed using an antibody-based detection assay, e.g., an ELISA, e.g.,as described in Example 12, e.g., relative to a reference composition(e.g., a vehicle control; an amino acid composition comprisingL-leucine, L-isoleucine, L-valine; an amino acid composition comprisingL-arginine, L-glutamine, and NAC; an amino acid composition comprisingL-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine;glutamine; arginine; isoleucine; leucine; or NAC).

In certain embodiments, the composition is capable of increasing, orincreases, expression of one or more collagen biomarkers (e.g.,procollagen lal) by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using primaryhepatic stellate cells, e.g., levels of procollagen lalin primaryhepatic stellate cells, e.g., as assessed using an antibody-baseddetection assay, e.g., an ELISA, e.g., as described in Example 12, e.g.,relative to a reference composition (e.g., a vehicle control; an aminoacid composition comprising L-leucine, L-isoleucine, L-valine; an aminoacid composition comprising L-arginine, L-glutamine, and NAC; an aminoacid composition comprising L-leucine, L-isoleucine, L-valine,L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine;leucine; or NAC).

In certain embodiments, the composition is capable of reducing, orreduces, hepatocyte inflammation by at least 5%, 10%, 15%, 20%, 20%,30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% asdetected using HepG2 cells, e.g., decreased activity, e.g., decreasedTNFα-induced activity of NF-kB in a reporter assay in HepG2 cells, asdescribed in Example 8, e.g., relative to a reference composition (e.g.,a vehicle control; an amino acid composition comprising L-leucine,L-isoleucine, L-valine; an amino acid composition comprising L-arginine,L-glutamine, and NAC; an amino acid composition comprising L-leucine,L-isoleucine, L-valine, L-arginine, and L-glutamine; or NAC).

In certain embodiments, the composition is capable of reducing, orreduces, TNFα-induced activity of NF-kB in HepG2 cells by at least 5%,10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, or 90% as detected using HepG2 cells, e.g., decreased activity,e.g., decreased TNFα-induced activity of NF-kB in a reporter assay inHepG2 cells, as described in Example 8, e.g., relative to a referencecomposition (e.g., a vehicle control; an amino acid compositioncomprising L-leucine, L-isoleucine, L-valine; an amino acid compositioncomprising L-arginine, L-glutamine, and NAC; an amino acid compositioncomprising L-leucine, L-isoleucine, L-valine, L-arginine, andL-glutamine; or NAC).

In certain embodiments, the composition is capable of increasing, orincreases, glucose tolerance, e.g., in a STAM mouse model or in a FATZOmouse model, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, asdetected using an assay of glucose levels, e.g., using glucose oxidase,e.g., using a glucometer, e.g., as described in Example 5, e.g.,relative to a reference composition (e.g., a vehicle control or apositive control, e.g., metformin).

In certain embodiments, the composition is capable of increasing, orincreases, blood glucose metabolism, e.g., in a STAM mouse model or in aFATZO mouse model, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or10%, as detected using an assay of glucose levels, e.g., using glucoseoxidase, e.g., using a glucometer, e.g., as described in Example 5,e.g., relative to a reference composition (e.g., a vehicle control or apositive control, e.g., metformin).

In certain embodiments, the composition is capable of decreasing, ordecreases, steatosis and/or inflammation by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2, e.g.,in primary hepatocytes, e.g., using an antibody-based detection assay,e.g., an ELISA, e.g., as described in Example 10, e.g., relative to areference composition (e.g., a vehicle control; an amino acidcomposition comprising L-leucine, L-isoleucine, L-valine; an amino acidcomposition comprising L-arginine, L-glutamine, and NAC; an amino acidcomposition comprising L-leucine, L-isoleucine, L-valine, L-arginine,and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; orNAC).

In certain embodiments, the composition is capable of decreasing, ordecreases, MCP1/CCL2 levels by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%,9%, or 10%, as detected using an assay of MCP1/CCL2, e.g., in primaryhepatocytes, e.g., using an antibody-based detection assay, e.g., anELISA, e.g., as described in Example 10, e.g., relative to a referencecomposition (e.g., a vehicle control; an amino acid compositioncomprising L-leucine, L-isoleucine, L-valine; an amino acid compositioncomprising L-arginine, L-glutamine, and NAC; an amino acid compositioncomprising L-leucine, L-isoleucine, L-valine, L-arginine, andL-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).In certain embodiments, the composition is capable of decreasing, ordecreases, TNFα inflammatory response by at least 1%, 2%, 3%, 4%, 5%,6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2 or anassay of IL-6, e.g., in primary hepatic stellate cells, e.g., using anantibody-based detection assay, e.g., an ELISA, e.g., as described inExample 11, e.g., relative to a reference composition (e.g., a vehiclecontrol; an amino acid composition comprising L-leucine, L-isoleucine,L-valine; an amino acid composition comprising L-arginine, L-glutamine,and NAC; an amino acid composition comprising L-leucine, L-isoleucine,L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine;isoleucine; leucine; or NAC).

In certain embodiments, the composition is capable of decreasing, ordecreases, MCP1/CCL2 levels and/or IL-6 levels by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2or an assay of IL-6, e.g., in primary hepatic stellate cells, e.g.,using an antibody-based detection assay, e.g., an ELISA, e.g., asdescribed in Example 11, e.g., relative to a reference composition(e.g., a vehicle control; an amino acid composition comprisingL-leucine, L-isoleucine, L-valine; an amino acid composition comprisingL-arginine, L-glutamine, and NAC; an amino acid composition comprisingL-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine;glutamine; arginine; isoleucine; leucine; or NAC).

In any of the foregoing embodiments, the reference composition comprisesa single amino acid entity, e.g., a L-amino acid entity, an I-amino acidentity, a V-amino acid entity, a R-amino acid entity, a Q-amino acidentity, or a NAC-amino acid entity, each assayed separately as a freeamino acid, or a combination of amino acid entities (e.g., a L-aminoacid entity, an I-amino acid entity, and a V-amino acid entity; aR-amino acid entity, a Q-amino acid entity, and a NAC-amino acid entity;a L-amino acid entity, an I-amino acid entity, V-amino acid entity, aR-amino acid entity, and a Q-amino acid entity). In certain embodiments,the reference composition comprises vehicle (e.g., PBS or saline).

In some embodiments, the composition that decreases and/or preventsliver fibrosis and/or liver injury comprises one or more branched-chainamino acid (BCAAs), one or more conditionally essential amino acid(CEAAs), and an antioxidant or reactive oxygen species (ROS) scavenger.

In some embodiments, the composition that decreases and/or preventshepatocyte inflammation comprises one or more BCAAs, one or more CEAAs,and an antioxidant or ROS scavenger.

In some embodiments, the composition that increases glucose tolerancecomprises one or more BCAAs, one or more CEAAs, and an antioxidant orROS scavenger.

In some embodiments, the composition that decreases and/or preventssteatosis comprises one or more BCAAs, one or more CEAAs, and anantioxidant or ROS scavenger.

In some embodiments, the composition that decreases and/or preventshepatocyte ballooning comprises one or more BCAAs, one or more CEAAs,and an antioxidant or ROS scavenger.

In an embodiment, the BCAA comprises a L-amino acid entity. In anembodiment, the BCAAs comprise a L-amino acid entity and an I-amino acidentity. In an embodiment, the BCAAs comprise a L-amino acid entity and aV-amino acid entity. In an embodiment, the BCAAs comprise a L-amino acidentity, a V-amino acid entity, and an I-amino acid entity. In anembodiment, the CEAA comprises a R-amino acid entity. In an embodiment,the CEAA comprises a Q-amino acid entity. In an embodiment, the CEAAcomprises a R-amino acid entity and a Q-amino acid entity. In anembodiment, the antioxidant or ROS scavenger comprises a NAC entity,e.g., NAC.

In some embodiments, the composition comprises a) a L-amino acid entity,an R-amino acid entity, and a Q-amino acid entity; and b) an antioxidantor ROS scavenger, e.g., a NAC entity, e.g., NAC.

In some embodiments, the composition further comprises an I-aminoacid-entity or a V-amino acid-entity. In other embodiments, thecomposition further comprises an I-amino acid-entity and a V-aminoacid-entity.

Production of the Amino Acid Compositions

Amino acids used to make the compositions may be agglomerated, and/orinstantized to aid in dispersal and/or solubilization.

The amino acid compositions of the present disclosure may be made usingamino acids and amino acid derivatives from the following sources, orother sources may used: FUSI-BCAA™ Instantized Blend (L-Leucine,L-Isoleucine and L-Valine in 2:1:1 weight ratio), FUSIL™ InstantizedL-Leucine, L-Arginine HCl, and L-Glutamine may be obtained fromAjinomoto Co., Inc; N-acetylcysteine may be obtained from SpectrumChemical.

To produce the amino acid compositions of the instant disclosure, thefollowing general steps may be used: the starting materials (individualamino acids and excipients) may be blended in a blending unit, followedby verification of blend uniformity and amino acid content, and fillingof the blended powder into stick packs or other unit dosage form. Thecontent of stick packs or other unit dosage forms may be dispersed inwater at time of use for oral administration.

Formulations

The pharmaceutical compositions of the present disclosure may be in aform suitable for oral use (for example as tablets, lozenges, hard orsoft capsules, aqueous or oily suspensions, emulsions, dispersiblepowders or granules, syrups or elixirs, medical food products,nutraceuticals), for topical use (for example as creams, ointments,gels, or aqueous or oily solutions or suspensions), for administrationby inhalation (for example as finely divided powder) or for parentaladministration (for example as a sterile aqueous or oily solution forintravenous, subcutaneous, intramuscular dosing or as a suppository forrectal dosing).

Excipients

The amino acid compositions of the present disclosure may be compoundedor formulated with one or more excipients. Non-limiting examples ofsuitable excipients include a tastant, a flavorant, a buffering agent, apreservative, a stabilizer, a binder, a compaction agent, a lubricant, adispersion enhancer, a disintegration agent, a flavoring agent, asweetener, and a coloring agent.

In some embodiments, the excipient comprises a buffering agent.Non-limiting examples of suitable buffering agents include citric acid,sodium citrate, magnesium carbonate, magnesium bicarbonate, calciumcarbonate, and calcium bicarbonate.

In some embodiments, the excipient comprises a preservative.Non-limiting examples of suitable preservatives include antioxidants,such as alpha-tocopherol and ascorbate, and antimicrobials, such asparabens, chlorobutanol, and phenol.

In some embodiments, the composition comprises a binder as an excipient.Non-limiting examples of suitable binders include starches,pregelatinized starches, gelatin, polyvinylpyrolidone, cellulose,methylcellulose, sodium carboxymethylcellulose, ethylcellulose,polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C12-C18 fattyacid alcohol, polyethylene glycol, polyols, saccharides,oligosaccharides, and combinations thereof.

In some embodiments, the composition comprises a lubricant as anexcipient. Non-limiting examples of suitable lubricants includemagnesium stearate, calcium stearate, zinc stearate, hydrogenatedvegetable oils, sterotex, polyoxyethylene monostearate, talc,polyethyleneglycol, sodium benzoate, sodium lauryl sulfate, magnesiumlauryl sulfate, and light mineral oil.

In some embodiments, the composition comprises a dispersion enhancer asan excipient. Non-limiting examples of suitable dispersants includestarch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, xanthangum, bentonite, purified wood cellulose, sodium starch glycolate,isoamorphous silicate, and microcrystalline cellulose as high HLBemulsifier surfactants.

In some embodiments, the composition comprises a disintegrant as anexcipient. In some embodiments, the disintegrant is a non-effervescentdisintegrant. Non-limiting examples of suitable non-effervescentdisintegrants include starches such as corn starch, potato starch,pregelatinized and modified starches thereof, sweeteners, clays, such asbentonite, microcrystalline cellulose, alginates, sodium starchglycolate, gums such as agar, guar, locust bean, karaya, pecitin, andtragacanth. In some embodiments, the disintegrant is an effervescentdisintegrant. Non-limiting examples of suitable effervescentdisintegrants include sodium bicarbonate in combination with citricacid, and sodium bicarbonate in combination with tartaric acid.

In some embodiments, the excipient comprises a flavoring agent.Flavoring agents can be chosen from synthetic flavor oils and flavoringaromatics; natural oils; extracts from plants, leaves, flowers, andfruits; and combinations thereof. In some embodiments, the flavoringagent is selected from cinnamon oils; oil of wintergreen; peppermintoils; clover oil; hay oil; anise oil; eucalyptus; vanilla; citrus oilsuch as lemon oil, orange oil, grape and grapefruit oil; and fruitessences including apple, peach, pear, strawberry, raspberry, cherry,plum, pineapple, and apricot.

In some embodiments, the excipient comprises a sweetener. Non-limitingexamples of suitable sweeteners include glucose (corn syrup), dextrose,invert sugar, fructose, and mixtures thereof (when not used as acarrier); saccharin and its various salts such as the sodium salt;dipeptide sweeteners such as aspartame; dihydrochalcone compounds,glycyrrhizin; Stevia Rebaudiana (Stevioside); chloro derivatives ofsucrose such as sucralose; and sugar alcohols such as sorbitol,mannitol, sylitol, and the like. Also contemplated are hydrogenatedstarch hydrolysates and the synthetic sweetener3,6-dihydro-6-methyl-1,2,3-oxathiazin-4-one-2,2-dioxide, particularlythe potassium salt (acesulfame-K), and sodium and calcium salts thereof.

In some embodiments, the composition comprises a coloring agent.Non-limiting examples of suitable color agents include food, drug andcosmetic colors (FD&C), drug and cosmetic colors (D&C), and externaldrug and cosmetic colors (Ext. D&C). The coloring agents can be used asdyes or their corresponding lakes.

Particular excipients may include one or more of: citric acid, lecithin,(e.g. Alcolec F100), sweeteners (e.g. sucralose, sucralose micronizedNF, acesulfame potassium (e.g. Ace-K)), a dispersion enhancer (e.g.xanthan gum (e.g. Ticaxan Rapid-3)), flavorings (e.g. vanilla custard#4306, Nat Orange WONF #1326, lime 865.0032U, and lemon 862.2169U), abitterness masking agent (e.g. 936.2160U), and natural or artificialcolorings (e.g. FD&C Yellow 6).

Methods of Treatment

The composition as described herein can be administered to improve liverfunction, e.g., in a patient with a liver disease. The composition asdescribed herein can also be administered to treat (e.g., reverse,reduce, ameliorate, or prevent) a disorder, e.g., a liver disease in asubject. The present disclosure provides methods of treating a liverdisease selected from fatty liver disease (steatohepatitis), alcoholicsteatohepatitis (ASH), non-alcoholic fatty liver disease (NAFLD),non-alcoholic fatty liver (NAFL), non-alcoholic steatohepatitis (NASH),liver fibrosis, and cirrhosis. In particular, an effective amount of thecomposition can be administered (e.g., according to a dosage regimendescribed herein) to treat a subject with non-alcoholic fatty liverdisease (NAFLD), non-alcoholic fatty liver (NAFL), non-alcoholicsteatohepatitis (NASH), or cirrhosis.

Patients with Liver Disease

In some embodiments, a subject has fatty liver disease selected fromNAFLD and AFLD. In some embodiments, the subject has pediatric NAFLD. Insome embodiments, the subject with NAFLD has NASH or NAFL. In someembodiments, the subject with AFLD has ASH.

In certain embodiments, the subject exhibits symptoms of gut leakiness.In certain embodiments, the subject has gut dysbiosis. In certainembodiments, the subject has gut microbiome disturbance. The subject mayhave increased levels of inflammatory cytokines, e.g., increased TNFα,relative to a normal subject without a fatty liver disease.

In certain embodiments, the subject exhibits muscle atrophy, e.g., has adecreased ratio of muscle tissue to adipose tissue, e.g., relative to anormal subject without a fatty liver disease. For example, the subjectexhibits muscle atrophy without fibrosis and/or cirrhosis.

In certain embodiments, the subject exhibits reverse lipid transportfrom adipose tissue to liver tissue.

In some embodiments, the subject has fibrosis. The subject may havecirrhosis. The subject may also have a metabolic syndrome.

In certain embodiments, the subject has one, two, or more (e.g., all) ofhepatocarcinoma, an increased risk of liver failure, or an increasedrisk of death.

In some embodiments, the subject has type 2 diabetes.

In some embodiments, the subject with a liver disease (e.g., NAFLD(e.g., NASH or NAFL) or AFLD (e.g., ASH)) is a mammal (e.g., a human).In some embodiments, the subject has been diagnosed with NAFLD, NASH orcirrhosis. In some embodiments, the subject has not received priortreatment with a composition as described herein (e.g., the subject is anaïve subject). In some embodiments, the subject with NAFLD, NASH orcirrhosis has diabetes (e.g., type 2 diabetes).

In some embodiments, the subject has NAFLD. In some embodiments, thesubject has NAFL. In certain embodiments, the subject (e.g., a child oran adolescent) has pediatric NAFLD. In some embodiments, the subject hashepatic steatosis. In some embodiments, a subject with pediatric NAFLDhas steatosis.

In some embodiments, the subject has non-alcoholic steatohepatitis(NASH). In some embodiments, the subject with NASH has fibrosis.

In some embodiments, the subject has cirrhosis. In some embodiments, thesubject with cirrhosis has fibrosis. In some embodiments, the subjectwith cirrhosis has hepatocarcinoma. In some embodiments, the subjectwith cirrhosis has an increased risk of liver failure. In someembodiments, the subject with cirrhosis has hepatocarcinoma, anincreased risk of liver failure, and an increased risk of death.

In some embodiments, a subject exhibits a symptom of liver disease (e.g.NAFLD, NASH, or cirrhosis), e.g., a metabolic symptom, prior toadministration of the composition. In some embodiments, a subjectexhibits a metabolic symptom of liver disease (e.g. NAFLD, NASH, orcirrhosis) selected from one, two, three, four, five, six, or more(e.g., all) of decreased fat metabolism, hepatocyte apoptosis,hepatocyte ballooning, inflammation of adipose tissue, inflammation ofhepatic tissue, hepatocyte ballooning, oxidative stress (e.g., reactiveoxygen species (ROS), decreased gut barrier function, decreased insulinsecretion, or decreased glucose tolerance (e.g., relative to a healthysubject without a liver disease).

In some embodiments, a subject exhibits modulated (e.g., increased)levels of a biomarker prior to administration of the composition. Insome embodiments, a subject exhibits modulated levels of a biomarkerselected from one, two, three, four, five, six, seven, eight, nine, ormore (e.g., all) of ACOX1; IL-10; NF-kB, an interferon, IL-2;glutathione (GSH); alanine aminotransferase (ALT); aspartateaminotransferase (AST); adiponectin; N-terminal fragment of type IIIcollagen (proC3); caspase-cleaved keratin 18 fragments (M30 and M65);IL-1β; C-reactive protein; PIIINP; TIMP1; MCP-1; or FGF-21 (e.g.,relative to a healthy subject without a liver disease).

In some embodiments, the subject exhibits increased levels of ALT, e.g.,relative to a healthy subject without a liver disease.

In some embodiments, the subject exhibits increased levels of AST, e.g.,relative to a healthy subject without a liver disease.

Improvement in Symptoms of Liver Disease

The composition as described herein can be administered to treat (e.g.,reverse, reduce, ameliorate, or prevent) a subject (e.g., a human) witha liver disease, thereby improving a symptom of a liver disease in thepatient. In some embodiments, the composition is administered to asubject with NAFLD. In some embodiments, the composition is administeredto a subject with NAFL. In some embodiments, the composition isadministered to a subject with NASH. In some embodiments, thecomposition is administered to a subject with cirrhosis of the liver.

In some embodiments, administration of a composition (e.g., at a dosageregimen described herein) results in an improvement in one or moresymptoms of NAFLD, e.g., a metabolic symptom of NAFLD, in a subject.

In some embodiments, administration of the composition results inincreased free fatty acid and lipid metabolism in a subject with NAFLD(e.g., a subject with pediatric NAFLD). In some embodiments,administration of the composition results in improved mitochondrialfunction in a subject with NAFLD (e.g., a subject with pediatric NAFLD).In some embodiments, administration of the composition results in whiteadipose tissue (WAT) browning in a subject with NAFLD (e.g., a subjectwith pediatric NAFLD).

In some embodiments, administration of the composition results indecreased reactive oxygen species (ROS) in a subject with NAFLD (e.g., asubject with pediatric NAFLD). In some embodiments, administration ofthe composition results in increased levels of glutathione (GSH) in asubject with NAFLD (e.g., a subject with pediatric NAFLD).

In some embodiments, administration of the composition results indecreased hepatic inflammation in a subject with NAFLD (e.g., a subjectwith pediatric NAFLD). In some embodiments, administration of thecomposition results in decreased hepatocyte ballooning in a subject withNAFLD (e.g., a subject with pediatric NAFLD).

In some embodiments, administration of the composition results inimproved gut barrier function in a subject with NAFLD (e.g., a subjectwith pediatric NAFLD).

In some embodiments, administration of the composition results inincreased insulin secretion in a subject with NAFLD (e.g., a subjectwith pediatric NAFLD). In some embodiments, administration of thecomposition results in improved glucose tolerance in a subject withNAFLD (e.g., a subject with pediatric NAFLD).

In some embodiments, the composition reduces or inhibits liver fibrosisin a subject with NAFLD (e.g., a subject with pediatric NAFLD). In someembodiments, the composition reduces or inhibits liver fibrosis in asubject with NAFLD (e.g., a subject with pediatric NAFLD).

In some embodiments, the composition reduces liver fat in a subject withNAFLD (e.g., a subject with pediatric NAFLD). In some embodiments, thecomposition reduces liver enzyme levels (e.g., ALT or AST) in blood orplasma from a subject with NAFLD (e.g., a subject with pediatric NAFLD).

In some embodiments, administration of a composition (e.g., at a dosageregimen described herein) including amino acid entities results in animprovement in one or more symptoms of NASH, e.g., a metabolic symptomof NASH, in a subject.

In some embodiments, administration of the composition results inincreased free fatty acid and lipid metabolism in a subject with NASH(e.g., a subject with NAFLD, fibrosis, and type 2 diabetes). In someembodiments, administration of the composition results in improvedmitochondrial function in a subject with NASH. In some embodiments,administration of the composition results in white adipose tissue (WAT)browning in a subject with NASH (e.g., a subject with NAFLD, fibrosis,and type 2 diabetes).

In some embodiments, administration of the composition results indecreased reactive oxygen species (ROS) in a subject with NASH (e.g., asubject with NAFLD, fibrosis, and type 2 diabetes). In some embodiments,administration of the composition results in increased levels ofglutathione (GSH) in a subject with NASH (e.g., a subject with NAFLD,fibrosis, and type 2 diabetes).

In some embodiments, administration of the composition results indecreased hepatic inflammation in a subject with NASH (e.g., a subjectwith NAFLD, fibrosis, and type 2 diabetes). In some embodiments,administration of the composition results in decreased hepatocyteballooning in a subject with NASH (e.g., a subject with NAFLD, fibrosis,and type 2 diabetes).

In some embodiments, administration of the composition results inimproved gut barrier function in a subject with NASH (e.g., a subjectwith NAFLD, fibrosis, and type 2 diabetes).

In some embodiments, administration of the composition results inincreased insulin secretion in a subject with NASH (e.g., a subject withNAFLD, fibrosis, and type 2 diabetes). In some embodiments,administration of the composition results in improved glucose tolerancein a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2diabetes).

In some embodiments, the composition reduces or inhibits liver fibrosisin a subject with NASH (e.g., a subject with NAFLD, fibrosis, and type 2diabetes). In some embodiments, the composition reduces or inhibitsliver fibrosis in a subject with NASH (e.g., a subject with NAFLD,fibrosis, and type 2 diabetes).

In some embodiments, the composition reduces liver fat in a subject withNASH (e.g., a subject with NAFLD, fibrosis, and type 2 diabetes). Insome embodiments, the composition reduces liver enzyme levels (e.g., ALTor AST) in blood or plasma from a subject with NASH (e.g., a subjectwith NAFLD, fibrosis, and type 2 diabetes).

In some embodiments, administration of a composition (e.g., at a dosageregimen described herein) including amino acid entities results in animprovement in one or more symptoms of cirrhosis, e.g., a metabolicsymptom of cirrhosis, in a subject.

In some embodiments, administration of the composition results indecreased reactive oxygen species (ROS) in a subject with cirrhosis(e.g., a subject with hepatocarcinoma, increased risk of liver failure,and increased risk of death). In some embodiments, administration of thecomposition results in increased levels of glutathione (GSH) in asubject with cirrhosis (e.g., a subject with hepatocarcinoma, increasedrisk of liver failure, and increased risk of death).

In some embodiments, administration of the composition results indecreased hepatic inflammation in a subject with cirrhosis (e.g., asubject with hepatocarcinoma, increased risk of liver failure, andincreased risk of death). In some embodiments, administration of thecomposition results in decreased hepatocyte ballooning in a subject withcirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liverfailure, and increased risk of death).

In some embodiments, administration of the composition results inimproved gut barrier function in a subject with cirrhosis (e.g., asubject with hepatocarcinoma, increased risk of liver failure, andincreased risk of death).

In some embodiments, administration of the composition results inincreased insulin secretion in a subject with cirrhosis (e.g., a subjectwith hepatocarcinoma, increased risk of liver failure, and increasedrisk of death). In some embodiments, administration of the compositionresults in improved glucose tolerance in a subject with cirrhosis (e.g.,a subject with hepatocarcinoma, increased risk of liver failure, andincreased risk of death).

In some embodiments, the composition reduces or inhibits liver fibrosisin a subject with cirrhosis (e.g., a subject with hepatocarcinoma,increased risk of liver failure, and increased risk of death). In someembodiments, the composition reduces or inhibits liver fibrosis in asubject with cirrhosis (e.g., a subject with hepatocarcinoma, increasedrisk of liver failure, and increased risk of death).

In some embodiments, the composition reduces liver fat in a subject withcirrhosis (e.g., a subject with hepatocarcinoma, increased risk of liverfailure, and increased risk of death). In some embodiments, thecomposition reduces liver enzyme levels (e.g., ALT or AST) in blood orplasma from a subject with cirrhosis (e.g., a subject withhepatocarcinoma, increased risk of liver failure, and increased risk ofdeath).

Dosage Regimens

The composition can be administered according to a dosage regimendescribed herein to treat (e.g., inhibit, reduce, ameliorate, orprevent) a disorder, e.g., a liver disease in a subject (e.g., a human).In some embodiments, the subject has NAFLD. In some embodiments, thesubject has NAFL. In some embodiments, the subject has NASH. In someembodiments, the subject has cirrhosis.

The composition can be provided to a patient with a liver disease (e.g.,NAFL, NASH, or cirrhosis) in either a single or multiple dosageregimens. In some embodiments, doses are administered, e.g., twicedaily, three times daily, four times daily, five times daily, six timesdaily, seven times daily, or more. In some embodiments, the compositionis administered for at least 2 days, 3 days, 4 days, 5 days, 6 days, 7days, or 2 weeks. In some embodiments, the composition is administeredfor at least 10 weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks,16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, or longer. In someembodiments, the composition is administered chronically, e.g., morethan 30 days, e.g., 31 days, 40 days, 50 days, 60 days, 3 months, 6months, 9 months, one year, two years, or three years).

In some embodiments, the composition is administered at a dose of about2 g to about 60 g total amino acids, e.g., once per day, twice per day,three times per day, four times per day, five times per day, or sixtimes per day (e.g., three times per day). In some embodiments, thecomposition is administered at a dose of about 5 g to about 15 g, about10 g to about 20 g, about 20 g to about 40 g, or about 30 g to about 50g total amino acids, e.g., once per day, twice per day, three times perday, four times per day, five times per day, or six times per day (e.g.,three times per day).

In some embodiments, the composition is administered at a dose of about5 g to about 10 g total amino acids, e.g., once per day, twice per day,three times per day, four times per day, five times per day, or sixtimes per day (e.g., three times per day). In some embodiments, thecomposition is administered at a dose of about 6 g total amino acids,e.g., once per day, twice per day, three times per day, four times perday, five times per day, or six times per day (e.g., three times perday). In an embodiment, the composition is administered at a dose ofabout 6 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about10 g to about 20 g total amino acids, e.g., once per day, twice per day,three times per day, four times per day, five times per day, or sixtimes per day (e.g., three times per day). In some embodiments, thecomposition is administered at a dose of about 12 g total amino acids,e.g., once per day, twice per day, three times per day, four times perday, five times per day, or six times per day (e.g., three times perday). In an embodiment, the composition is administered at a dose ofabout 12 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about20 g to about 40 g total amino acids, e.g., once per day, twice per day,three times per day, four times per day, five times per day, or sixtimes per day (e.g., three times per day). In some embodiments, thecomposition is administered at a dose of about 18 g total amino acids,e.g., once per day, twice per day, three times per day, four times perday, five times per day, or six times per day (e.g., three times perday). In an embodiment, the composition is administered at a dose ofabout 18 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about20 g to about 40 g total amino acids, e.g., once per day, twice per day,three times per day, four times per day, five times per day, or sixtimes per day (e.g., three times per day). In some embodiments, thecomposition is administered at a dose of about 24 g total amino acids,e.g., once per day, twice per day, three times per day, four times perday, five times per day, or six times per day (e.g., three times perday). In an embodiment, the composition is administered at a dose ofabout 24 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about30 g to about 50 g total amino acids, e.g., once per day, twice per day,three times per day, four times per day, five times per day, or sixtimes per day (e.g., three times per day). In some embodiments, thecomposition is administered at a dose of about 48 g total amino acids,e.g., once per day, twice per day, three times per day, four times perday, five times per day, or six times per day (e.g., three times perday). In an embodiment, the composition is administered at a dose ofabout 48 g total amino acids three times per day.

In some embodiments, the composition is administered at a dose of about5 grams, about 8 grams, about 9 grams, about 10 grams, about 11 grams,about 12 grams, about 13 grams, about 14 grams, about 15 grams, about 16grams, about 17 grams, about 18 grams, about 19 about grams, about 20grams, about 21 grams, about 22 grams, about 24 grams, about 25 grams,about 26 grams, about 27 grams, about 28 grams, about 29 grams, or about30 grams total amino acids (e.g., about 12 g or about 24 g), e.g., onceper day, twice per day, three times per day, four times per day, fivetimes per day, or six times per day (e.g., three times per day).

In some embodiments, the composition is administered every 2 hours,every 3 hours, every 4 hours, every 5 hours, every 6 hours, every 7hours, every 8 hours, every 9 hours, or every 10 hours to a subject witha liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD (e.g., ASH)).

In an embodiment, the composition is administered to a subject withNAFLD prior to a meal. In an embodiment, the composition is administeredto a subject with NAFLD concurrent with a meal. In an embodiment, thecomposition is administered to a subject with NAFLD following a meal.

In an embodiment, the composition is administered to a subject with NAFLprior to a meal. In an embodiment, the composition is administered to asubject with NAFL concurrent with a meal. In an embodiment, thecomposition is administered to a subject with NAFL following a meal.

In an embodiment, the composition is administered to a subject with NASHprior to a meal. In an embodiment, the composition is administered to asubject with NASH concurrent with a meal. In an embodiment, thecomposition is administered to a subject with NASH following a meal.

In an embodiment, the composition is administered to the subject withcirrhosis prior to a meal. In an embodiment, the composition isadministered to a subject with cirrhosis concurrent with a meal. In anembodiment, the composition is administered to a subject with cirrhosisfollowing a meal.

In an embodiment, the composition includes at least 1 g of leucine, atleast 0.5 g of isoleucine, at least 0.5 g of valine, at least 1.5 g ofarginine (or 1.81 g of arginine HCl), at least 2 g of glutamine, and atleast 0.15 g of N-acetylcysteine for administration three times per day(e.g., for a total of at least 18 g per day).

In an embodiment, the composition includes about 1 g of leucine, about0.5 g of isoleucine, about 0.5 g of valine, about 1.5 g of arginine (or1.81 g of arginine HCl), about 2 g of glutamine, and about 0.15 g ofN-acetylcysteine for administration three times per day (e.g., for atotal of about 18 g per day).

In an embodiment, the composition includes at least 2 g of leucine, atleast 1 g of isoleucine, at least 1 g of valine, at least 3.0 g ofarginine (or 3.62 g of arginine HCl), at least 4 g of glutamine, and atleast 0.3 g of N-acetylcysteine for administration three times per day(e.g., a total of at least 36 g per day).

In an embodiment, the composition includes about 2 g of leucine, about 1g of isoleucine, about 1 g of valine, about 3.0 g or arginine (or 3.62 gof arginine HCl), about 4 g of glutamine, and about 0.3 g ofN-acetylcysteine for administration three times per day (e.g., a totalof about 36 g per day).

In an embodiment, the composition includes at least 4 g of leucine, atleast 2 g of isoleucine, at least 2 g of valine, at least 6.0 g ofarginine (or 7.24 g of arginine HCl), at least 8 g of glutamine, and atleast 0.6 g of N-acetylcysteine for administration three times per day(e.g., a total of at least 72 g per day).

In an embodiment, the composition includes about 4 g of leucine, about 2g of isoleucine, about 2 g of valine, about 6.0 g of arginine (or 7.24 gof arginine HCl), about 8 g of glutamine, and about 0.6 g ofN-acetylcysteine for administration three times per day (e.g., a totalof about 72 g per day).

In an embodiment, the composition includes at least 1 g of leucine, atleast 0.5 g of isoleucine, at least 0.5 g of valine, at least 0.75 g ofarginine (or 0.905 g of arginine HCl), at least 2 g of glutamine, and atleast 0.15 g of N-acetylcysteine for administration three times per day(e.g., for a total of at least 18 g per day).

In an embodiment, the composition includes about 1 g of leucine, about0.5 g of isoleucine, about 0.5 g of valine, about 0.75 g of arginine (or0.905 g of arginine HCl), about 2 g of glutamine, and about 0.15 g ofN-acetylcysteine for administration three times per day (e.g., for atotal of about 18 g per day).

In an embodiment, the composition includes at least 2 g of leucine, atleast 1 g of isoleucine, at least 1 g of valine, at least 1.5 g ofarginine (or 1.81 g of arginine HCl), at least 4 g of glutamine, and atleast 0.3 g of N-acetylcysteine for administration three times per day(e.g., a total of at least 36 g per day).

In an embodiment, the composition includes about 2 g of leucine, about 1g of isoleucine, about 1 g of valine, about 1.5 g or arginine (or 1.81 gof arginine HCl), about 4 g of glutamine, and about 0.3 g ofN-acetylcysteine for administration three times per day (e.g., a totalof about 36 g per day).

In an embodiment, the composition includes at least 4 g of leucine, atleast 2 g of isoleucine, at least 2 g of valine, at least 3.0 g ofarginine (or 3.62 g of arginine HCl), at least 8 g of glutamine, and atleast 0.6 g of N-acetylcysteine for administration three times per day(e.g., a total of at least 72 g per day).

In an embodiment, the composition includes about 4 g of leucine, about 2g of isoleucine, about 2 g of valine, about 3.0 g of arginine (or 3.62 gof arginine HCl), about 8 g of glutamine, and about 0.6 g ofN-acetylcysteine for administration three times per day (e.g., a totalof about 72 g per day).

In an embodiment, the composition includes at least 1 g of leucine, atleast 0.5 g of isoleucine, at least 0.25 g of valine, at least 0.75 g ofarginine (or 0.905 g of arginine HCl), at least 1 g of glutamine, and atleast 0.225 g of N-acetylcysteine for administration three times per day(e.g., for a total of at least 18 g per day).

In an embodiment, the composition includes about 1 g of leucine, about0.5 g of isoleucine, about 0.25 g of valine, about 0.75 g of arginine(or 0.905 g of arginine HCl), about 1 g of glutamine, and about 0.225 gof N-acetylcysteine for administration three times per day (e.g., for atotal of about 18 g per day).

In an embodiment, the composition includes at least 2 g of leucine, atleast 1 g of isoleucine, at least 0.5 g of valine, at least 1.5 g ofarginine (or 1.81 g of arginine HCl), at least 2 g of glutamine, and atleast 0.45 g of N-acetylcysteine for administration three times per day(e.g., a total of at least 36 g per day).

In an embodiment, the composition includes about 2 g of leucine, about 1g of isoleucine, about 0.5 g of valine, about 1.5 g or arginine (or 1.81g of arginine HCl), about 2 g of glutamine, and about 0.45 g ofN-acetylcysteine for administration three times per day (e.g., a totalof about 36 g per day).

In an embodiment, the composition includes at least 4 g of leucine, atleast 2 g of isoleucine, at least 1 g of valine, at least 3 g ofarginine (or 3.62 g of arginine HCl), at least 4 g of glutamine, and atleast 0.9 g of N-acetylcysteine for administration three times per day(e.g., a total of at least 72 g per day).

In an embodiment, the composition includes about 4 g of leucine, about 2g of isoleucine, about 1 g of valine, about 3 g of arginine (or 3.62 gof arginine HCl), about 4 g of glutamine, and about 0.9 g ofN-acetylcysteine for administration three times per day (e.g., a totalof about 72 g per day).

In an embodiment, the composition includes at least 1 g of leucine, atleast 0.5 g of isoleucine, at least 0.25 g of valine, at least 0.75 g ofarginine (or 0.905 g of arginine HCl), at least 1 g of glutamine, atleast 0.225 g of N-acetylcysteine, and at least 1.5 g or about 1.67 g ofserine for administration three times per day (e.g., for a total of atleast 18 g per day or for a total of at least 20 g per day).

In an embodiment, the composition includes about 1 g of leucine, about0.5 g of isoleucine, about 0.25 g of valine, about 0.75 g of arginine(or 0.905 g of arginine HCl), about 1 g of glutamine, about 0.225 g ofN-acetylcysteine, and about 1.5 g or about 1.67 g of serine foradministration three times per day (e.g., for a total of about 18 g perday or for a total of at least 20 g per day).

In an embodiment, the composition includes at least 2 g of leucine, atleast 1 g of isoleucine, at least 0.5 g of valine, at least 1.5 g ofarginine (or 1.81 g of arginine HCl), at least 2 g of glutamine, atleast 0.45 g of N-acetylcysteine, and at least 3 g or about 3.33 g ofserine for administration three times per day (e.g., a total of at least36 g per day or for a total of at least 40 g per day).

In an embodiment, the composition includes about 2 g of leucine, about 1g of isoleucine, about 0.5 g of valine, about 1.5 g or arginine (or 1.81g of arginine HCl), about 2 g of glutamine, about 0.45 g ofN-acetylcysteine, and about 3 g or about 3.33 g of serine foradministration three times per day (e.g., a total of about 36 g per dayor for a total of at least 40 g per day).

In an embodiment, the composition includes at least 4 g of leucine, atleast 2 g of isoleucine, at least 1 g of valine, at least 3 g ofarginine (or 3.62 g of arginine HCl), at least 4 g of glutamine, atleast 0.9 g of N-acetylcysteine, and at least 6 g or about 6.67 g ofserine for administration three times per day (e.g., a total of at least90 g per day).

In an embodiment, the composition includes about 4 g of leucine, about 2g of isoleucine, about 1 g of valine, about 3 g of arginine (or 3.62 gof arginine HCl), about 4 g of glutamine, about 0.9 g ofN-acetylcysteine, and about 6 g or about 6.67 g of serine foradministration three times per day (e.g., a total of about 90 g perday). In some embodiments, the composition comprises four stick packs,each stick pack comprising 25% of the quantity of each amino acidincluded in the composition (e.g., as described herein).

Secondary Agents

In some embodiments, the method further comprises administering afarnesoid X receptor (FXR) agonist, a stearoyl CoA desaturase inhibitor,a CCR2 and CCR5 chemokine antagonist, a PPAR alpha and delta agonist, acaspase inhibitor, a galectin-3 inhibitor, an acetyl CoA carboxylaseinhibitor, or an ileal sodium bile acid co-transporter inhibitor priorto, concurrently with, or after administration of the amino acidcomposition.

In some embodiments, the method further includes administering an FXRagonist. In some embodiments, the FXR agonist is obeticholic acid. Insome embodiments, the method further includes administering one or moreof: LMB-763, LJN-452, emricasan, and cenicriviroc.

Dietary Compositions

The composition including amino acid entities can be dietarycompositions, e.g., chosen from a medical food, a functional food, or asupplement.

The composition including amino acid entities can be for use as adietary composition, e.g., chosen from a medical food, a functionalfood, or a supplement. In some embodiments, the dietary composition isfor use in a method comprising adminstering the composition to asubject.

In some embodiments, the subject has one or both of type 2 diabetes or arelatively high BMI.

In some embodiments, the subject has fatty liver disease.

In some embodiments, the subject has NAFLD (e.g., pediatric NAFLD). Inan embodiment, the subject has NASH. In an embodiment, the subject hasNAFL.

In some embodiments, the subject has AFLD. In an embodiment, the subjecthas ASH.

In some embodiments, the subject has one, two, three, four, or more(e.g., all) of fibrosis, cirrhosis, hepatocarcinoma, an increased riskof liver failure, or an increased risk of death.

In some embodiments, the composition promotes weight loss in thesubject.

In some embodiments, administration of the dietary composition resultsin an improvement in one or more metabolic symptoms in the subject,e.g., one or more metabolic symptoms is selected from the following:increased free fatty acid and lipid metabolism (e.g., in the liver),improved mitochondrial function, white adipose tissue (WAT) browning,decreased reactive oxygen species (ROS), increased levels of glutathione(GSH), decreased hepatic inflammation, decreased hepatocyte ballooning,improved gut barrier function, increased insulin secretion, or glucosetolerance. In certain embodiments, administration of the compositionresults in an improvement in one or more metabolic symptoms after atreatment period of 24 hours.

The method can further include determining the level of one, two, three,four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen,fourteen, or more (e.g., all) of the following:

a) alanine aminotransferase (ALT);

b) aspartate aminotransferase (AST);

c) adiponectin;

d) N-terminal fragment of type III collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

f) IL-1 beta;

g) C-reactive protein;

h) PIIINP;

i) a tissue inhibitor of metalloproteinase (TIMP); e.g., TIMP1 or TIMP2;

j) MCP-1;

k) FGF-21;

l) Col1a1;

m) Acta2;

n) a matrix metalloproteinase (MMP), e.g., MMP-13, MMP-2, MMP-9,MT1-MMP, MMP-3, or MMP-10;

o) ACOX1;

p) IL-10; or

q) NF-kB.

In certain embodiments, administration of the composition results in animprovement in one or more of a)-q) after a treatment period of 24hours.

In some embodiments, the subject exhibits increased levels of one orboth of ALT or AST prior to administration of the composition, e.g.,relative to a healthy subject without a liver disease. In someembodiments, administration of the composition results in a decrease inlevels of one or both of ALT or AST.

Methods of Providing an Amino Acid to a Subject

The present disclosure features a method of providing amino acidentities to a subject comprising administering to the subject aneffective amount of a composition described herein, e.g., a compositioncomprising a leucine (L)-amino acid entity, a arginine (R)-amino acidentity, a glutamine (Q)-amino acid entity; and an antioxidant orreactive oxygen species (ROS) scavenger, e.g., a N-acetylcysteine (NAC)entity, e.g., NAC. In some embodiments, at least one amino acid entityis not a peptide of more than 20 amino acid residues in length.

The present disclosure also features a method of increasing one, two,three, or more (e.g., all) amino acid entities in a subject comprisingadministering to the subject an effective amount of the compositiondescribed herein. In some embodiments, administration of the compositionresults in an increase in the amino acid entities in one, two, or more(e.g., all) of blood, plasma, or serum of the subject, e.g., in a blood,plasma, or serum sample from the subject.

Biomarkers

Any of the methods disclosed herein can include evaluating or monitoringthe effectiveness of administering a composition including amino acidentities to a subject with a liver disease (e.g., NAFLD (e.g., NASH orNAFL) or AFLD (e.g., ASH)).

In embodiments, the value of effectiveness to the composition intreating a subject with a liver disease (e.g., NAFLD (e.g., NASH orNAFL) or AFLD (e.g., ASH)) comprises a measure of one, two, three, four,five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen,fifteen, sixteen, or more (e.g., all) of the following:

a) alanine aminotransferase (ALT);

b) aspartate aminotransferase (AST);

c) adiponectin;

d) N-terminal fragment of type III collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

f) IL-1 beta;

g) C-reactive protein;

h) PIIINP;

i) a tissue inhibitor of metalloproteinase (TIMP); e.g., TIMP1 or TIMP2;

j) MCP-1;

k) FGF-21;

l) Col1a1;

m) Acta2;

n) a matrix metalloproteinase (MMP), e.g., MMP-13, MMP-2, MMP-9,MT1-MMP, MMP-3, or MMP-10;

o) ACOX1;

p) IL-10; or

q) NF-kB.

In some embodiments of any of the methods disclosed herein, the measureof one or more of a)-q) is obtained from a sample acquired from thesubject with a liver disease (e.g., NAFLD (e.g., NASH or NAFL) or AFLD(e.g., ASH)). In some embodiments, the sample is chosen from a bloodsample (e.g., a plasma sample) or a liver sample.

In some embodiments, the subject is evaluated prior to receiving,during, or after receiving, a composition including amino acid entities.

In some embodiments, administration of the composition including aminoacid entities (e.g., at a dose of about 2 g to about 60 g total aminoacids, e.g., about 6 g, about 12 g, about 18 g, about 24 g, or about 48g), results in an improvement in one, two, three, four, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, or more (e.g., all) of the following:

a) alanine aminotransferase (ALT);

b) aspartate aminotransferase (AST);

c) adiponectin;

d) N-terminal fragment of type III collagen (proC3);

e) caspase-cleaved keratin 18 fragments (M30 and M65);

f) IL-1 beta;

g) C-reactive protein;

h) PIIINP;

i) a tissue inhibitor of metalloproteinase (TIMP); e.g., TIMP1 or TIMP2;

j) MCP-1;

k) FGF-21;

l) Col1a1;

m) Acta2;

n) a matrix metalloproteinase (MMP), e.g., MMP-13, MMP-2, MMP-9,MT1-MMP, MMP-3, or MMP-10;

o) ACOX1;

p) IL-10; or

q) NF-kB.

In some embodiments, administration of the composition including aminoacid entities (e.g., at a dose of about 2 g to about 60 g total aminoacids, e.g., about 6 g, about 12 g, about 18 g, about 24 g, or about 48g three times daily), results in an improvement in one, two, three,four, five, six, seven, eight, nine, ten, or more (e.g., all) of a)-k)after a treatment period of, about 24 hours, about 72 hours, about 1week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about6 weeks, about 7 weeks, about 8 weeks, about 9 weeks, about 10 weeks,about 11 weeks, or 12 weeks. In certain embodiments, administration ofthe composition results in an improvement in one, two, three, four,five, or more (e.g., all) of a)-k) after a treatment period of about 2weeks.

Numbered Embodiments

The invention is further described with reference to the followingnumbered embodiments.

1. A composition comprising:

a) a leucine (L)-amino acid entity, an arginine (R)-amino acid entity,and a glutamine (Q)-amino acid entity; and

b) an antioxidant or reactive oxygen species (ROS) scavenger, e.g., aN-acetylcysteine (NAC) entity, e.g., NAC;

provided that:

c) at least one amino acid entity is not provided as a peptide of morethan 20 amino acid residues in length, and optionally wherein:

(i) an amino acid entity (e.g., at least one, two, or three of the aminoacid entities) of (a) is selected from Table 2; or

(ii) (A) one or both of the R-amino acid entity and the Q-amino acidentity are present at a higher amount (wt. %) than the L-amino acidentity, or (B) the composition further comprises a serine (S)-amino acidentity.

1A. The composition of embodiment 1, wherein the composition satisfiesthe property of (i).

1B. The composition of any of the preceding embodiments, wherein thecomposition satisfies the property of (ii)(A).

1C. The composition of any of the preceding embodiments, wherein thecomposition satisfies the property of (ii)(B).

1D. The composition of any of the preceding embodiments, wherein thecomposition further comprises an S-amino acid entity, and wherein theS-amino acid entity is present at a higher amount than any other aminoacid entity.

2. The composition of any of embodiments 1-1D, wherein the compositioncomprises an amino acid and three amino acid entities.

3. The composition of any of embodiments 1-1D, wherein the compositioncomprises an amino acid precursor and three amino acid entities.

4. The composition of any of embodiments 1-1D, wherein the compositioncomprises an amino acid metabolite and three amino acid entities.

5. The composition of any of embodiments 1-1D, wherein the compositioncomprises an amino acid derivative and three amino acid entities.

6. The composition of any of embodiments 1-1D, wherein the compositioncomprises two amino acids and two amino acid entities.

7. The composition of any of embodiments 1-1D, wherein the compositioncomprises two amino acid precursors and two amino acid entities.

8. The composition of any of embodiments 1-1D, wherein the compositioncomprises two amino acid metabolites and two amino acid entities.

9. The composition of any of embodiments 1-1D, wherein the compositioncomprises two amino acid derivatives and two amino acid entities.

10. The composition of any of embodiments 1-1D, wherein the compositioncomprises three amino acids and one amino acid entity.

11. The composition of any of embodiments 1-1D, wherein the compositioncomprises three amino acid precursors and one amino acid entity.

12. The composition of any of embodiments 1-1D, wherein the compositioncomprises three amino acid metabolites and one amino acid entity.

13. The composition of any of embodiments 1-1D, wherein the compositioncomprises three amino acid derivatives and one amino acid entity.

14. The composition of any of embodiments 1-2, wherein the compositioncomprises L-leucine, a R-amino acid entity, and a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

15. The composition of any of embodiments 1-2, 2, 14, or 380, whereinthe composition comprises L-leucine, R-arginine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

16. The composition of any of embodiments 1-2, 14, or 381, wherein thecomposition comprises L-leucine, argininosuccinate, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

17. The composition of any of embodiments 1-2, 14, or 382, wherein thecomposition comprises L-leucine, citrulline, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

18. The composition of any of embodiments 1-2, 14, or 383, wherein thecomposition comprises L-leucine, aspartate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

19. The composition of any of embodiments 1-2, 14, or 384, wherein thecomposition comprises L-leucine, L-glutamate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

20. The composition of any of embodiments 1-2, 14, or 385, wherein thecomposition comprises L-leucine, ornithine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

21. The composition of any of embodiments 1-2, 14, or 386, wherein thecomposition comprises a L-leucine, agmatine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

22. The composition of any of embodiments 1-2, 14, or 387, wherein thecomposition comprises a L-leucine, creatine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

23. The composition of any of embodiments 1-2, 14, or 388, wherein thecomposition comprises L-leucine, D-arginine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

24. The composition of any of embodiments 1-2, 14, or 389, wherein thecomposition comprises L-leucine, N-acetyl-arginine, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

25. The composition of any of embodiments 1-2, 14, or 428, wherein thecomposition comprises L-leucine, a R-amino acid entity, L-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

26. The composition of any of embodiments 1-2, 14, or 429, wherein thecomposition comprises L-leucine, a R-amino acid entity, glutamate, andan antioxidant or ROS scavenger, e.g., a NAC entity.

27. The composition of any of embodiments 1-2, 14, or 430, wherein thecomposition comprises L-leucine, a R-amino acid entity, carbamoyl-P, andan antioxidant or ROS scavenger, e.g., a NAC entity.

28. The composition of any of embodiments 1-2, 14, or 431, wherein thecomposition comprises L-leucine, a R-amino acid entity, D-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

29. The composition of any of embodiments 1-2, 14, or 432, wherein thecomposition comprises L-leucine, a R-amino acid entity,N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NACentity.

30. The composition of any of embodiments 1-2, 14, or 445, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and NAC.

31. The composition of any of embodiments 1-2, 14, or 446, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and serine.

32. The composition of any of embodiments 1-2, 14, or 447, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and acetylserine.

33. The composition of any of embodiments 1-2, 14, or 448, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and cystathionine.

34. The composition of any of embodiments 1-2, 14, or 449, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and glutathione.

35. The composition of any of embodiments 1-2, 14, or 450, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and homocysteine.

36. The composition of any of embodiments 1-2, 14, or 451, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and methionine.

37. The composition of any of embodiments 1-2, 14, or 452, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and D-cysteine.

38. The composition of any of embodiments 1-2, 14, or 453, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and L-cysteine.

39. The composition of any of embodiments 1-2, 14, or 454, wherein thecomposition comprises L-leucine, a R-amino acid entity, a Q-amino acidentity, and cystine.

40. The composition of any of embodiments 1-2, 14, 380, or 428, whereinthe composition comprises L-leucine, L-arginine, L-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

41. The composition of any of embodiments 1-2, 14, 381, or 429, whereinthe composition comprises L-leucine, argininosuccinate, glutamate, andan antioxidant or ROS scavenger, e.g., a NAC entity.

42. The composition of any of embodiments 1-2, 14, 382, or 431, whereinthe composition comprises L-leucine, citrulline, D-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

43. The composition of any of embodiments 1-2, 14, or 383, wherein thecomposition comprises L-leucine, aspartate, N-acetyl-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

44. The composition of any of embodiments 1-2, 14, 380, or 445, whereinthe composition comprises L-leucine, L-arginine, a Q-amino acid entity,and NAC.

45. The composition of any of embodiments 1-2, 14, 381, or 446, whereinthe composition comprises L-leucine, argininosuccinate, a Q-amino acidentity, and serine.

46. The composition of any of embodiments 1-2, 14, 382, or 447, whereinthe composition comprises L-leucine, citrulline, a Q-amino acid entity,and acetylserine.

47. The composition of any of embodiments 1-2, 14, 383, or 448, whereinthe composition comprises L-leucine, aspartate, a Q-amino acid entity,and cystathionine.

48. The composition of any of embodiments 1-2, 14, 384, or 449, whereinthe composition comprises L-leucine, glutamate, a Q-amino acid entity,and glutathione.

49. The composition of any of embodiments 1-2, 14, 385, or 450, whereinthe composition comprises L-leucine, ornithine, a Q-amino acid entity,and homocysteine.

50. The composition of any of embodiments 1-2, 14, 386, or 451, whereinthe composition comprises L-leucine, agmatine, a Q-amino acid entity,and methionine.

51. The composition of any of embodiments 1-2, 14, 387, or 452, whereinthe composition comprises L-leucine, creatine, a Q-amino acid entity,and D-cysteine.

52. The composition of any of embodiments 1-2, 14, 388, or 453, whereinthe composition comprises L-leucine, D-arginine, a Q-amino acid entity,and L-cysteine.

53. The composition of any of embodiments 1-2, 14, 389, or 454, whereinthe composition comprises L-leucine, N-acetyl-arginine, a Q-amino acidentity, and cystine.

54. The composition of any of embodiments 1-2, 14, 428, or 445, whereinthe composition comprises L-leucine, a R-amino acid entity, L-glutamine,and NAC.

55. The composition of any of embodiments 1-2, 14, 429, or 446, whereinthe composition comprises L-leucine, a R-amino acid entity, glutamate,and serine.

56. The composition of any of embodiments 1-2, 14, 430, or 447, whereinthe composition comprises L-leucine, a R-amino acid entity, carbamoyl-P,and acetylserine.

57. The composition of any of embodiments 1-2, 14, 432, or 448, whereinthe composition comprises L-leucine, a R-amino acid entity,N-acetyl-glutamine, and cystathionine.

58. The composition of any of embodiments 1-2, 14, 433, or 449, whereinthe composition comprises L-leucine, a R-amino acid entity, L-glutamine,and glutathione.

59. The composition of any of embodiments 1-2, 14, or 450, wherein thecomposition comprises L-leucine, a R-amino acid entity, glutamate, andhomocysteine.

60. The composition of any of embodiments 1-2, 14, or 451, wherein thecomposition comprises L-leucine, a R-amino acid entity, carbamoyl-P, andmethionine.

61. The composition of any of embodiments 1-2, 14, or 452, wherein thecomposition comprises L-leucine, a R-amino acid entity,N-acetyl-glutamine, and D-cysteine.

62. The composition of any of embodiments 1-2, 14, or 453, wherein thecomposition comprises L-leucine, a R-amino acid entity, L-glutamine, andL-cysteine.

63. The composition of any of embodiments 1-2, 14, or 454, wherein thecomposition comprises L-leucine, a R-amino acid entity, a glutamate, andcystine.

64. The composition of any of embodiments 1-2, 14, 380, or 445, whereinthe composition comprises L-leucine, L-arginine, L-glutamine, and NAC.

65. The composition of any of embodiments 1-2, 14, 381, or 446, whereinthe composition comprises L-leucine, argininosuccinate, glutamate, andserine.

66. The composition of any of embodiments 1-2, 14, 382, or 447, whereinthe composition comprises L-leucine, citrulline, carbamoyl-P, andacetylserine.

67. The composition of any of embodiments 1-2, 14, 383, or 448, whereinthe composition comprises L-leucine, aspartate, D-glutamine, andcystathionine.

68. The composition of any of embodiments 1-2, 14, 384, or 449, whereinthe composition comprises L-leucine, glutamate, L-glutamine, andglutathione.

69. The composition of any of embodiments 1-2, 14, 385, or 450, whereinthe composition comprises L-leucine, ornithine, glutamate, andhomocysteine.

70. The composition of any of embodiments 1-2, 14, 386, or 451, whereinthe composition comprises L-leucine, agmatine, carbamoyl-P, andmethionine.

71. The composition of any of embodiments 1-2, 14, 387, or 452, whereinthe composition comprises L-leucine, creatine, D-glutamine andD-cysteine.

72. The composition of any of embodiments 1-2, 14, 388, or 453, whereinthe composition comprises L-leucine, D-arginine, a Q-amino acid entity,and L-cysteine.

73. The composition of any of embodiments 1-2, 14, 389, or 454, whereinthe composition comprises L-leucine, N-acetyl-arginine,argininosuccinate, and cystine.

74. The composition of embodiment 1 or 3, wherein the compositioncomprises oxo-leucine, a R-amino acid entity, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

75. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, and a Q-aminoacid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

76. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, L-arginine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

77. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, argininosuccinate, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

78. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, citrulline, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

79. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, aspartate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

80. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, glutamate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

81. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, ornithine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

82. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, agmatine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

83. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, creatine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

84. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, D-arginine, a Q-amino acid entity,and an antioxidant or ROS scavenge, e.g., a NAC entity.

85. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, N-acetyl-arginine, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

86. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, L-glutamine,and an antioxidant or ROS scavenger, e.g., a NAC entity.

87. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, glutamate, andan antioxidant or ROS scavenger, e.g., a NAC entity.

88. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, carbamoyl-P,and an antioxidant or ROS scavenger, e.g., a NAC entity.

89. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, D-glutamine,and an antioxidant or ROS scavenger, e.g., a NAC entity.

90. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity,N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NACentity.

91. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and NAC.

92. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and serine.

93. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and acetylserine.

94. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and cystathionine.

95. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and glutathione.

96. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and homocysteine.

97. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and methionine.

98. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and D-cysteine.

99. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and L-cysteine.

100. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and a NAC entity.

101. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a Q-amino acidentity, and cystine.

102. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, L-arginine, L-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

103. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, argininosuccinate, glutamate, and anantioxidant or ROS scavenger, e.g., a NAC entity.

104. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, citrulline, D-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

105. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, aspartate, N-acetyl-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

106. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, L-arginine, a Q-amino acid entity,and NAC.

107. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, argininosuccinate, a Q-amino acidentity, and serine.

108. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, citrulline, a Q-amino acid entity,and acetylserine.

109. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, aspartate, a Q-amino acid entity, andcystathionine.

110. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, glutamate, a Q-amino acid entity, andglutathione.

111. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, ornithine, a Q-amino acid entity, andhomocysteine.

112. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, agmatine, a Q-amino acid entity, andmethionine.

113. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, creatine, a Q-amino acid entity, andD-cysteine.

114. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, D-arginine, a Q-amino acid entity,and L-cysteine.

115. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, N-acetyl-arginine, a Q-amino acidentity, and cystine.

116. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, L-glutamine,and NAC.

117. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, glutamate, andserine.

118. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, carbamoyl-P,and acetylserine.

119. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity,N-acetyl-glutamine, and cystathionine.

120. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, L-glutamine,and glutathione.

121. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, glutamate, andhomocysteine.

122. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, carbamoyl-P,and methionine.

123. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity,N-acetyl-glutamine, and D-cysteine.

124. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, L-glutamine,and L-cysteine.

125. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, a R-amino acid entity, a glutamate,and cystine.

126. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, L-arginine, L-glutamine, and NAC.

127. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, argininosuccinate, glutamate, andserine.

128. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, citrulline, carbamoyl-P, andacetylserine.

129. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, aspartate, D-glutamine, andcystathionine.

130. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, N-acetyl-glutamine, L-glutamine, andglutathione.

131. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, ornithine, glutamate, andhomocysteine.

132. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, agmatine, carbamoyl-P, andmethionine.

133. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, creatine, D-glutamine and D-cysteine.

134. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, D-arginine, a Q-amino acid entity,and L-cysteine.

135. The composition of embodiment 1-1D, 3, or 74, wherein thecomposition comprises oxo-leucine, N-acetyl-arginine, argininosuccinate,and cystine.

136. The composition of embodiment 1-1D or 4, wherein the compositioncomprises HMB, a R-amino acid entity, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

137. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, L-arginine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

138. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, argininosuccinate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

139. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, citrulline, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

140. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, aspartate, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

141. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, glutamate, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

142. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, ornithine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

143. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, agmatine, a Q-amino acid entity, and anantioxidant or ROS scavenger e.g., a NAC entity.

144. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, creatine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

145. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, D-arginine, a Q-amino acid entity, and anantioxidant or ROS scavenge e.g., a NAC entity.

146. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, N-acetyl-arginine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

147. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, L-glutamine, and anantioxidant or ROS scavenger, e.g., an antioxidant, e.g., a NAC entity.

148. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, glutamate, and anantioxidant or ROS scavenger, e.g., an antioxidant, e.g., a NAC entity.

149. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, carbamoyl-P, and anantioxidant or ROS scavenger, e.g., an antioxidant, e.g., a NAC entity.

150. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, D-glutamine, and anantioxidant or ROS scavenger, e.g., an antioxidant, e.g., a NAC entity.

151. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, N-acetyl-glutamine,and an antioxidant or ROS scavenger, e.g., an antioxidant, e.g., a NACentity.

152. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and NAC.

153. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and serine.

154. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and acetylserine.

155. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and cystathionine.

156. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and glutathione.

157. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and homocysteine.

158. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and methionine.

159. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and D-cysteine.

160. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and L-cysteine.

161. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and cysteine.

162. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, a Q-amino acid entity,and cystine.

163. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, L-arginine, L-glutamine, and an antioxidantor ROS scavenger, e.g., a NAC entity.

164. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, argininosuccinate, glutamate, and anantioxidant or ROS scavenger, e.g., a NAC entity.

165. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, citrulline, D-glutamine, and an antioxidantor ROS scavenger, e.g., a NAC entity.

166. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, aspartate, N-acetyl-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

167. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, L-arginine, a Q-amino acid entity, and NAC.

168. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, argininosuccinate, a Q-amino acid entity, andserine.

169. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, citrulline, a Q-amino acid entity, andacetylserine.

170. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, aspartate, a Q-amino acid entity, andcystathionine.

171. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, glutamate, a Q-amino acid entity, andglutathione.

172. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, ornithine, a Q-amino acid entity, andhomocysteine.

173. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, agmatine, a Q-amino acid entity, andmethionine.

174. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, creatine, a Q-amino acid entity, andD-cysteine.

175. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, D-arginine, a Q-amino acid entity, andL-cysteine.

176. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, N-acetyl-arginine, a Q-amino acid entity, andcystine.

177. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, L-glutamine, and NAC.

178. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, glutamate, and serine.

179. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, carbamoyl-P, andacetylserine.

180. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, N-acetyl-glutamine,and cystathionine.

181. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, L-glutamine, andglutathione.

182. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, glutamate, andhomocysteine.

183. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, carbamoyl-P, andmethionine.

184. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, N-acetyl-glutamine,and D-cysteine.

185. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, a R-amino acid entity, L-glutamine, andL-cysteine. 186. The composition of embodiment 1-1D, 4, or 136, whereinthe composition comprises HMB, a R-amino acid entity, a glutamate, andcystine.

187. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, L-arginine, L-glutamine, and NAC.

188. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, argininosuccinate, glutamate, and serine.

189. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, citrulline, carbamoyl-P, and acetylserine.

190. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, aspartate, D-glutamine, and cystathionine.

191. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, N-acetyl-glutamine, L-glutamine, andglutathione.

192. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, ornithine, glutamate, and homocysteine.

193. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, agmatine, carbamoyl-P, and methionine.

194. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, creatine, D-glutamine and D-cysteine.

195. The composition of embodiment 1-1D, 4, or 136, wherein thecomposition comprises HMB, D-arginine, a Q-amino acid entity, andL-cysteine. 196. The composition of embodiment 1-1D, 4, or 136, whereinthe composition comprises HMB, N-acetyl-arginine, argininosuccinate, andcystine.

197. The composition of embodiment 1-1D, or 4, wherein the compositioncomprises isovaleryl-CoA, a R-amino acid entity, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

198. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, L-arginine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

199. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, argininosuccinate, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

200. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, citrulline, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

201. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, aspartate, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

202. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, glutamate, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

203. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, ornithine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

204. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, agmatine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

205. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, creatine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

206. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, D-arginine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

207. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, N-acetyl-arginine, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

208. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

209. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, glutamate,and an antioxidant or ROS scavenger, e.g., a NAC entity.

210. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,carbamoyl-P, and an antioxidant or ROS scavenger, e.g., a NAC entity.

211. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

212. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NACentity.

213. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and NAC.

214. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and serine.

215. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and acetylserine.

216. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and cystathionine.

217. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and glutathione.

218. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and homocysteine.

219. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and methionine.

220. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and D-cysteine.

221. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and L-cysteine.

222. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and cysteine.

223. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, a Q-aminoacid entity, and cystine.

224. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, L-arginine, L-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

225. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, argininosuccinate, glutamate, andan antioxidant or ROS scavenger, e.g., a NAC entity.

226. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, citrulline, D-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

227. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, aspartate, N-acetyl-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

228. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, L-arginine, a Q-amino acid entity,and NAC.

229. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, argininosuccinate, a Q-amino acidentity, and serine.

230. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, citrulline, a Q-amino acid entity,and acetylserine.

231. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, aspartate, a Q-amino acid entity,and cystathionine.

232. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, glutamate, a Q-amino acid entity,and glutathione.

233. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, ornithine, a Q-amino acid entity,and homocysteine.

234. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, agmatine, a Q-amino acid entity,and methionine.

235. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, creatine, a Q-amino acid entity,and D-cysteine.

236. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, D-arginine, a Q-amino acid entity,and L-cysteine.

237. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, N-acetyl-arginine, a Q-amino acidentity, and cystine.

238. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,L-glutamine, and NAC.

239. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, glutamate,and serine.

240. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,carbamoyl-P, and acetylserine.

241. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,N-acetyl-glutamine, and cystathionine.

242. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,L-glutamine, and glutathione.

243. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, glutamate,and homocysteine.

244. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,carbamoyl-P, and methionine.

245. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,N-acetyl-glutamine, and D-cysteine.

246. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity,L-glutamine, and L-cysteine.

247. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, a R-amino acid entity, aglutamate, and cystine.

248. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, L-arginine, L-glutamine, and NAC.

249. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, argininosuccinate, glutamate, andserine.

250. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, citrulline, carbamoyl-P, andacetylserine.

251. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, aspartate, D-glutamine, andcystathionine.

252. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, N-acetyl-glutamine, L-glutamine,and glutathione.

253. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, ornithine, glutamate, andhomocysteine.

254. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, agmatine, carbamoyl-P, andmethionine.

255. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, creatine, D-glutamine andD-cysteine.

256. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, D-arginine, a Q-amino acid entity,and L-cysteine.

257. The composition of embodiment 1-1D, 4, or 197, wherein thecomposition comprises isovaleryl-CoA, N-acetyl-arginine,argininosuccinate, and cystine.

258. The composition of embodiment 1-1D or 5, wherein the compositioncomprises D-leucine, a R-amino acid entity, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

259. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, L-arginine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

260. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, argininosuccinate, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

261. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, citrulline, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

262. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, aspartate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

263. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, glutamate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

264. The composition of embodiment 1-1D, 5, or 258, wherein compositioncomprises D-leucine, ornithine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

265. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, agmatine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

266. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, creatine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

267. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, D-arginine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

268. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, N-acetyl-arginine, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

269. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, L-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

270. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, glutamate, andan antioxidant or ROS scavenger, e.g., a NAC entity.

271. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, carbamoyl-P, andan antioxidant or ROS scavenger, e.g., a NAC entity.

272. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, D-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

273. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity,N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NACentity.

274. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and NAC.

275. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and serine.

276. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and acetylserine.

277. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and cystathionine.

278. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and glutathione.

279. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and homocysteine.

280. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and methionine.

281. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and D-cysteine.

282. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and L-cysteine.

283. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and cysteine.

284. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a Q-amino acidentity, and cystine.

285. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, L-arginine, L-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

286. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, argininosuccinate, glutamate, and anantioxidant or ROS scavenger e.g., a NAC entity.

287. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, citrulline, D-glutamine, and anantioxidant or ROS scavenger e.g., a NAC entity.

288. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, aspartate, N-acetyl-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

289. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, L-arginine, a Q-amino acid entity, andNAC.

290. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, argininosuccinate, a Q-amino acidentity, and serine.

291. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, citrulline, a Q-amino acid entity, andacetylserine.

292. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, aspartate, a Q-amino acid entity, andcystathionine.

293. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, glutamate, a Q-amino acid entity, andglutathione.

294. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, ornithine, a Q-amino acid entity, andhomocysteine.

295. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, agmatine, a Q-amino acid entity, andmethionine.

296. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, creatine, a Q-amino acid entity, andD-cysteine.

297. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, D-arginine, a Q-amino acid entity, andL-cysteine.

298. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, N-acetyl-arginine, a Q-amino acidentity, and cystine.

299. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, L-glutamine, andNAC.

300. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, glutamate, andserine.

301. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, carbamoyl-P, andacetylserine.

302. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity,N-acetyl-glutamine, and cystathionine.

303. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, L-glutamine, andglutathione.

304. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, glutamate, andhomocysteine.

305. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, carbamoyl-P, andmethionine.

306. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity,N-acetyl-glutamine, and D-cysteine.

307. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, L-glutamine, andL-cysteine.

308. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, a R-amino acid entity, a glutamate, andcystine.

309. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, L-arginine, L-glutamine, and NAC.

310. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, argininosuccinate, glutamate, andserine.

311. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, citrulline, carbamoyl-P, andacetylserine.

312. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, aspartate, D-glutamine, andcystathionine.

313. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, N-acetyl-glutamine, L-glutamine, andglutathione.

314. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, ornithine, glutamate, and homocysteine.

315. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, agmatine, carbamoyl-P, and methionine.

316. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, creatine, D-glutamine and D-cysteine.

317. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, D-arginine, a Q-amino acid entity, andL-cysteine.

318. The composition of embodiment 1-1D, 5, or 258, wherein thecomposition comprises D-leucine, N-acetyl-arginine, argininosuccinate,and cystine.

319. The composition of embodiment 1-1D or 5, wherein the compositioncomprises N-acetyl-leucine, a R-amino acid entity, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

320. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, L-arginine, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

321. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, argininosuccinate, a Q-aminoacid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

322. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, citrulline, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

323. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, aspartate, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

324. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, glutamate, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

325. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, ornithine, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

326. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, agmatine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

327. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, creatine, a Q-amino acid entity,and an antioxidant or ROS scavenger, e.g., a NAC entity.

328. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, D-arginine, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

329. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, N-acetyl-arginine, a Q-aminoacid entity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

330. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,L-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

331. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

332. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,carbamoyl-P, and an antioxidant or ROS scavenger, e.g., a NAC entity.

333. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,D-glutamine, and an antioxidant or ROS scavenger, e.g., a NAC entity.

334. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NACentity.

335. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and NAC.

336. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and serine.

337. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and acetylserine.

338. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and cystathionine.

339. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and glutathione.

340. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and homocysteine.

341. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and methionine.

342. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and D-cysteine.

343. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and L-cysteine.

344. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and cysteine.

345. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, a Q-aminoacid entity, and cystine.

346. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, L-arginine, L-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

347. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, argininosuccinate, glutamate,and an antioxidant or ROS scavenger, e.g., a NAC entity.

348. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, citrulline, D-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

349. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, aspartate, N-acetyl-glutamine,and an antioxidant or ROS scavenger, e.g., a NAC entity.

350. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, L-arginine, a Q-amino acidentity, and NAC.

351. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, argininosuccinate, a Q-aminoacid entity, and serine.

352. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, citrulline, a Q-amino acidentity, and acetylserine.

353. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, aspartate, a Q-amino acidentity, and cystathionine.

354. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, glutamate, a Q-amino acidentity, and glutathione.

355. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, ornithine, a Q-amino acidentity, and homocysteine.

356. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, agmatine, a Q-amino acid entity,and methionine.

357. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, creatine, a Q-amino acid entity,and D-cysteine.

358. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, D-arginine, a Q-amino acidentity, and L-cysteine.

359. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, N-acetyl-arginine, a Q-aminoacid entity, and cystine.

360. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,L-glutamine, and NAC.

361. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,glutamate, and serine.

362. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,carbamoyl-P, and acetylserine.

363. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,N-acetyl-glutamine, and cystathionine.

364. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,L-glutamine, and glutathione.

365. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,glutamate, and homocysteine.

366. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,carbamoyl-P, and methionine.

367. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,N-acetyl-glutamine, and D-cysteine.

368. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity,L-glutamine, and L-cysteine.

369. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, a R-amino acid entity, aglutamate, and cystine.

370. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, L-arginine, L-glutamine, andNAC.

371. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, argininosuccinate, glutamate,and serine.

372. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, citrulline, carbamoyl-P, andacetylserine.

373. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, aspartate, D-glutamine, andcystathionine.

374. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, N-acetyl-glutamine, L-glutamine,and glutathione.

375. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, ornithine, glutamate, andhomocysteine.

376. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, agmatine, carbamoyl-P, andmethionine.

377. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, creatine, D-glutamine andD-cysteine.

378. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, D-arginine, a Q-amino acidentity, and L-cysteine.

379. The composition of embodiment 1-1D, 5, or 319, wherein thecomposition comprises N-acetyl-leucine, N-acetyl-arginine,argininosuccinate, and cystine.

380. The composition of embodiment 1-1D or 2, wherein the compositioncomprises a L-amino acid entity, L-arginine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

381. The composition of embodiment 1-1D or 2, wherein the compositioncomprises a L-amino acid entity, argininosuccinate, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

382. The composition of embodiment 1-1D, 3, or 4, wherein thecomposition comprises a L-amino acid entity, citrulline, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

383. The composition of embodiment 1-1D or 3, wherein the compositioncomprises a L-amino acid entity, aspartate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

384. The composition of embodiment 1-1D or 3, wherein the compositioncomprises a L-amino acid entity, glutamate, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

385. The composition of embodiment 1-1D or 4, wherein the compositioncomprises a L-amino acid entity, ornithine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

386. The composition of embodiment 1-1D or 4, wherein the compositioncomprises a L-amino acid entity, agmatine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

387. The composition of embodiment 1-1D or 4, wherein the compositioncomprises a L-amino acid entity, creatine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

388. The composition of embodiment 1-1D or 5, wherein the compositioncomprises a L-amino acid entity, D-arginine, a Q-amino acid entity, andan antioxidant or ROS scavenger, e.g., a NAC entity.

389. The composition of embodiment 1-1D or 5, wherein the compositioncomprises a L-amino acid entity, N-acetyl-arginine, a Q-amino acidentity, and an antioxidant or ROS scavenger, e.g., a NAC entity.

390. The composition of embodiment 1-1D, 3, or 384, wherein thecomposition comprises L-leucine, glutamate, L-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

391. The composition of embodiment 1-1D, 4, or 385, wherein thecomposition comprises L-leucine, ornithine, L-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

392. The composition of embodiment 1-1D, 4, or 386, wherein thecomposition comprises a L-amino acid entity, agmatine, L-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

393. The composition of embodiment 1-1D, 4, or 387, wherein thecomposition comprises a L-amino acid entity, creatine, L-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

394. The composition of embodiment 1-1D, 4, or 388, wherein thecomposition comprises a L-amino acid entity, D-arginine, L-glutamine,and an antioxidant or ROS scavenger, e.g., a NAC entity.

395. The composition of embodiment 1-1D, 4, or 389, wherein thecomposition comprises a L-amino acid entity, D-arginine,N-acetyl-arginine, and an antioxidant or ROS scavenger, e.g., a NACentity.

396. The composition of embodiment 1-1D or 380, wherein the compositioncomprises a L-amino acid entity, L-arginine, L-glutamine, and anantioxidant or ROS scavenger, e.g., a NAC entity.

397. The composition of any of embodiments 1-2, or 381, wherein thecomposition comprises a L-amino acid entity, argininosuccinate,glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

398. The composition of embodiment 1-1D, 3, 4, or 382, wherein thecomposition comprises a L-amino acid entity, citrulline, carbamoyl-P,and an antioxidant or ROS scavenger, e.g., a NAC entity.

399. The composition of embodiment 1-1D, 3, or 383, wherein thecomposition comprises a L-amino acid entity, aspartate, glutamate, andan antioxidant or ROS scavenger, e.g., a NAC entity.

400. The composition of embodiment 1-1D, 3, or 384, wherein thecomposition comprises a L-amino acid entity, glutamate, D-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

401. The composition of embodiment 1-1D, 4, or 385, wherein thecomposition comprises a L-amino acid entity, ornithine,N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NACentity.

402. The composition of embodiment 1-1D, 4, or 386, wherein thecomposition comprises a L-amino acid entity, agmatine, L-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

403. The composition of embodiment 1-1D, 4, or 387, wherein thecomposition comprises a L-amino acid entity, creatine, glutamate, and anantioxidant or ROS scavenger, e.g., a NAC entity.

404. The composition of embodiment 1-1D, 5, or 388, wherein thecomposition comprises a L-amino acid entity, D-arginine, carbamoyl-P,and an antioxidant or ROS scavenger, e.g., a NAC entity.

405. The composition of embodiment 1-1D, 5, or 389, wherein thecomposition comprises a L-amino acid entity, N-acetyl-arginine,glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

406. The composition of embodiment 1-1D, 3, 80, or 445, wherein thecomposition comprises a L-amino acid entity, L-arginine, L-glutamine,and NAC.

407. The composition of any of embodiments 1-2, 381, or 446, wherein thecomposition comprises a L-amino acid entity, argininosuccinate,glutamate, and serine.

408. The composition of embodiment 1-1D, 3, 4, 382, or 447, wherein thecomposition comprises a L-amino acid entity, citrulline, carbamoyl-P,and acetylserine.

409. The composition of embodiment 1-1D, 3, 383, or 448, wherein thecomposition comprises a L-amino acid entity, aspartate, glutamate, andcystathionine.

410. The composition of embodiment 1-1D, 3, 384, or 449, wherein thecomposition comprises a L-amino acid entity, glutamate, D-glutamine, andglutathione.

411. The composition of embodiment 1-1D, 4, 385, or 448, wherein thecomposition comprises a L-amino acid entity, ornithine,N-acetyl-glutamine, and cystathionine.

412. The composition of embodiment 1-1D, 4, 386, or 450, wherein thecomposition comprises a L-amino acid entity, agmatine, L-glutamine, andhomocysteine.

413. The composition of embodiment 1-1D, 4, 387, or 451, wherein thecomposition comprises a L-amino acid entity, creatine, glutamate, andmethionine.

414. The composition of embodiment 1-1D, 5, 388, or 454, wherein thecomposition comprises a L-amino acid entity, D-arginine, carbamoyl-P,and D-cysteine.

415. The composition of embodiment 1-1D, 5, 389, or 453, wherein thecomposition comprises a L-amino acid entity, N-acetyl-arginine,glutamate, and L-cysteine.

416. The composition of embodiment 1-1D, 380, or 454, wherein thecomposition comprises a L-amino acid entity, L-arginine, L-glutamine,and cystine.

417. The composition of embodiment 1-1D, 6, or 445, wherein thecomposition comprises a L-amino acid entity, L-arginine, a Q-amino acid,and NAC.

418. The composition of any of embodiments 1-2, or 446, wherein thecomposition comprises a L-amino acid entity, argininosuccinate, aQ-amino acid, and serine.

419. The composition of embodiment 1-1D, 3, or 447, wherein thecomposition comprises a L-amino acid entity, citrulline, a Q-amino acid,and acetylserine.

420. The composition of embodiment 1-1D, 4, or 448, wherein thecomposition comprises a L-amino acid entity, aspartate, a Q-amino acid,and cystathionine.

421. The composition of embodiment 1-1D, 3, or 449, wherein thecomposition comprises a L-amino acid entity, glutamate, a Q-amino acid,and glutathione.

422. The composition of embodiment 1-1D, 4, or 448, wherein thecomposition comprises a L-amino acid entity, ornithine, a Q-amino acid,and cystathionine.

423. The composition of embodiment 1-1D, 4, or 450, wherein thecomposition comprises a L-amino acid entity, agmatine, a Q-amino acid,and homocysteine.

424. The composition of embodiment 1-1D, 4, or 451, wherein thecomposition comprises a L-amino acid entity, creatine, a Q-amino acid,and methionine.

425. The composition of embodiment 1-1D, 5, or 452, wherein thecomposition comprises a L-amino acid entity, D-arginine, a Q-amino acid,and D-cysteine.

426. The composition of embodiment 1-1D, 5, or 453, wherein thecomposition comprises a L-amino acid entity, N-acetyl-arginine, aQ-amino acid, and L-cysteine.

427. The composition of embodiment 1-1D, 5, or 454, wherein thecomposition comprises a L-amino acid entity, L-arginine, a Q-amino acid,and cystine.

428. The composition of embodiment 1-1D or 2, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, L-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

429. The composition of embodiment 1-1D, 3, or 4, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,glutamate, and an antioxidant or ROS scavenger, e.g., a NAC entity.

430. The composition of embodiment 1-1D or 4, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, carbamoyl-P, andan antioxidant or ROS scavenger, e.g., a NAC entity.

431. The composition of embodiment 1-1D or 5, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, D-glutamine, andan antioxidant or ROS scavenger, e.g., a NAC entity.

432. The composition of embodiment 1-1D or 5, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity,N-acetyl-glutamine, and an antioxidant or ROS scavenger, e.g., a NACentity.

433. The composition of embodiment 1-1D, 5, or 431, wherein thecomposition comprises a L-leucine, a R-amino acid entity, D-glutamine,and an antioxidant or ROS scavenger, e.g., a NAC entity.

434. The composition of embodiment 1-1D, 4 or 430, wherein thecomposition comprises a L-leucine, L-arginine, carbamoyl-P, and anantioxidant or ROS scavenger, e.g., a NAC entity.

435. The composition of any of embodiments 1-2, 428, or 445, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,L-glutamine, and NAC.

436. The composition of embodiment 1-1D, 4, 429, or 446, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,glutamate, and serine.

437. The composition of embodiment 1-1D, 4, 430, or 447, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,carbamoyl-P, and acetylserine.

438. The composition of embodiment 1-1D, 5, 431, or 448, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,D-glutamine, and cystathionine.

439. The composition of embodiment 1-1D, 5, 432, or 449, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,N-acetyl-glutamine, and glutathione.

440. The composition of any of embodiments 1-2, 428, or 450, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,L-glutamine, and homocysteine.

441. The composition of embodiment 1-1D, 3, 4, 429, or 451, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,glutamate, and methionine.

442. The composition of embodiment 1-1D, 4, 430, or 452, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,carbamoyl-P, and D-cysteine

443. The composition of embodiment 1-1D, 5, 431, or 453, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,D-glutamine, and L-cysteine.

444. The composition of embodiment 1-1D, 5, 432, or 454, wherein thecomposition comprises a L-amino acid entity, a R-amino acid entity,N-acetyl-glutamine, and cystine.

445. The composition of embodiment 1-1D or 5, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and NAC.

446. The composition of embodiment 1-1D or 3, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and serine.

447. The composition of embodiment 1-1D or 3, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and acetylserine.

448. The composition of embodiment 1-1D or 3, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and cystathionine.

449. The composition of embodiment 1-1D or 4, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and glutathione.

450. The composition of embodiment 1-1D or 4, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and homocysteine.

451. The composition of embodiment 1-1D or 4, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and methionine.

452. The composition of embodiment 1-1D or 5, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and D-cysteine.

453. The composition of embodiment 1-1D or 5, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and L-cysteine.

454. The composition of embodiment 1-1D or 5, wherein the compositioncomprises a L-amino acid entity, a R-amino acid entity, a Q-amino acidentity, and cystine.

455. The composition of embodiment 1-1D or 2, wherein the compositioncomprises a L-amino acid, ornithine, a Q-amino acid entity, and anantioxidant or ROS scavenger, e.g., a NAC entity.

456. The composition of embodiment 1-1D or 455, wherein the compositioncomprises L-leucine, ornithine, 1-glutamine, and NAC.

457. The composition of embodiment 1-1D or 455, wherein the compositioncomprises HMB, ornithine, 1-glutamine, and NAC.

458. The composition of any of the foregoing embodiments, wherein thecomposition comprises L-leucine or a leucine metabolite (e.g., HMB),L-arginine or an L-arginine metabolite (e.g., creatine), 1-glutamine,and NAC or a NAC metabolite, e.g., glutathione.

459. The composition of any of the foregoing embodiments, wherein thecomposition comprises L-leucine or a leucine metabolite (e.g., HMB),L-arginine or an L-arginine metabolite (e.g., creatine), L-glutamine,and NAC or a NAC metabolite, e.g., glutathione.

460. The composition of any of the previous embodiments, furthercomprising an isoleucine (I)-amino acid entity.

461. The composition of embodiment 460, wherein the I-amino acid entityis an amino acid.

462. The composition of embodiment 460 or 461, wherein the amino acidentity is L-isoleucine.

463. The composition of embodiment 460, wherein the I-amino acid entityis an amino acid precursor.

464. The composition of embodiment 460 or 463, wherein the I-amino acidentity is 2-oxo-3-methyl-valerate.

465. The composition of embodiment 460 or 463, wherein the I-amino acidentity is threonine.

466. The composition of embodiment 460, wherein the I-amino acid entityis an amino acid metabolite.

467. The composition of embodiment 460 or 466, wherein the I-amino acidentity is 2-oxo-3-methyl-valerate

468. The composition of embodiment 460 or 466, wherein the I-amino acidentity is methylbutyrl-CoA.

469. The composition of embodiment 460, wherein the I-amino acid entityis an amino acid derivative.

470. The composition of embodiment 460 or 469, wherein the I-amino acidentity is D-isoleucine.

471. The composition of embodiment 460 or 469, wherein the I-amino acidentity is N-acetyl-isoleucine.

472. The composition of any of the previous embodiments, furthercomprising a valine (V)-amino acid entity.

473. The composition of embodiment 472, wherein the V-amino acid entityis an amino acid.

474. The composition of embodiment 472 or 473, wherein the V-amino acidentity is L-valine.

475. The composition of embodiment 472, wherein the V-amino acid entityis an amino acid precursor.

476. The composition of embodiment 472 or 475, wherein the V-amino acidentity is 2-oxo-valerate.

477. The composition of embodiment 472, wherein the V-amino acid entityis an amino acid metabolite.

478. The composition of embodiment 472 or 477, wherein the V-amino acidentity is isobutryl-CoA.

479. The composition of embodiment 472 or 477, wherein the V-amino acidentity is 3-HIB-CoA.

480. The composition of embodiment 472 or 477, wherein the V-amino acidentity is 3-HIB.

481. The composition of embodiment 472, wherein the V-amino acid entityis an amino acid derivative.

482. The composition of embodiment 472 or 481, wherein the V-amino acidentity is D-valine.

483. The composition of embodiment 472 or 481, wherein the V-amino acidentity is N-acetyl-valine.

484. The composition of any of the preceding embodiments, furthercomprising L-glycine.

485. The composition of any of the preceding embodiments, furthercomprising an S-amino acid entity (e.g., L-serine, phosphoserine,P-hydroxypyruvate, L-glycine, tryptophan, acetylserine, cystathionine,phosphatidylserine, or any combination thereof, e.g., L-serine andL-glycine).

486. The composition of any of the preceding embodiments, furthercomprising carnitine.

487. The composition of any of the preceding embodiments, comprising:

a) a L-amino acid entity chosen from L-leucine or a salt thereof, orβ-hydroxy-β-methybutyrate (HMB) or a salt thereof or a combination ofL-leucine or a salt thereof and HMB ora salt thereof;

b) an R-amino acid entity chosen from L-arginine or a salt thereof,ornithine or a salt thereof, or creatine or a salt thereof or acombination of two or three of L-arginine or a salt thereof, ornithineor a salt thereof, or creatine or a salt thereof;

c) L-glutamine or a salt thereof; and

d) N-acetylcysteine (NAC) or a salt thereof.

488. The composition of any of the preceding embodiments, wherein theL-Leucine is provided as part of a dipeptide comprising L-Leucine, or asalt thereof, or a tripeptide comprising L-Leucine, or a salt thereof.

489. The composition of any of the preceding embodiments, wherein theL-Arginine is provided as part of a dipeptide comprising L-Arginine, ora salt thereof, or a tripeptide comprising L-Arginine, or a saltthereof.

490. The composition of any of the preceding embodiments, wherein theL-Glutamine is provided as part of a dipeptide comprising L-Glutamine,or a salt thereof, or a tripeptide comprising L-Glutamine, or a saltthereof

491. The composition of any of the preceding embodiments, wherein theNAC is provided as a part of a dipeptide comprising NAC, or a saltthereof, or a tripeptide comprising NAC, or a salt thereof.

492. The composition of any of the preceding embodiments, wherein one,two, three, or four of methionine (M), tryptophan (W), valine (V), orcysteine (C) is absent, or if present, is present at less than 10 weight(wt.) % of the composition.

493. The composition of any of the preceding embodiments, wherein thetotal wt. % of (a)-(d) is greater than the total wt. % of any otheramino acid entity in the composition.

494. The composition of any of the preceding embodiments, wherein one,two, three, or four of the amino acids in (a)-(d) is provided as part ofa dipeptide or tripeptide, e.g., in an amount of at least 10 wt. % ofthe composition.

495. The composition of embodiment 494, wherein the dipeptide is ahomodipeptide or heterodipeptide of any of the amino acids in (a)-(d),e.g., one, two, three, or four of the amino acids in (a)-(d) is ahomodipeptide or heterodipeptide.

496. The composition of embodiment 494, wherein the tripeptide is ahomotripeptide or heterotripeptide of any of the amino acids in (a)-(d),e.g., one, two, three, or four of the amino acids in (a)-(d) is ahomotripeptide or heterotripeptide.

497. The composition of any of the preceding embodiments, wherein (a) isa L-amino acid entity dipeptide or a salt thereof (e.g., a L-leucinedipeptide or a salt thereof)

498. The composition of embodiment 497, wherein (a) is a homodipeptideor a heterodipeptide, e.g., Ala-Leu.

499. The composition of any of the preceding embodiments, wherein (b) isa L-arginine dipeptide or a salt thereof.

500. The composition of embodiment 499, wherein (b) is a homodipeptideor a heterodipeptide, e.g., Ala-Arg.

501. The composition of any of the preceding embodiments, wherein (c) isa L-glutamine dipeptide or a salt thereof.

502. The composition of embodiment 501, wherein (c) is a homodipeptide,e.g., Gln-Gln, or wherein (c) is a heterodipeptide, e.g., Ala-Gln.

503. The composition of any of the preceding embodiments, wherein:

f) a wt. % of the L-glutamine or a salt thereof in the composition isgreater than the wt. % of the R-amino acid entity;

g) the wt. % of the L-glutamine or a salt thereof in the composition isgreater than the wt. % of the L-amino acid entity;

h) the wt. % of the R-amino acid entity in the composition is greaterthan the wt. % of the L-amino acid entity; or

i) a combination of two or three of (f)-(h).

504. The composition of any of the preceding embodiments, wherein thewt. % of the L-glutamine or a salt thereof in the composition is atleast 5% greater than the wt. % of the R-amino acid entity, e.g., thewt. % of the L-glutamine or a salt thereof is at least 10%, 15%, 20%, or25% greater than the wt. % of the R-amino acid entity

505. The composition of any of the preceding embodiments, wherein thewt. % of the L-glutamine or a salt thereof in the composition is atleast 20% greater than the wt. % of the L-amino acid entity, e.g., thewt. % of the L-glutamine or a salt thereof in the composition is atleast 25%, 30%, 35%, 40%, 45%, or 50% greater than the wt. % of theL-amino acid entity.

506. The composition of any of the preceding embodiments, wherein thewt. % of the R-amino acid entity in the composition is at least 10%greater than the wt. % of the L-amino acid entity, e.g., the wt. % ofthe R-amino acid entity in the composition is at least 15%, 20%, 25%, or30% greater than the wt. % of the L-amino acid entity.

507. The composition of any of the preceding embodiments, wherein:

j) the ratio of the L-amino acid entity to the R-amino acid entity is atleast 1:4, or at least 2:5, and not more than 3:4, e.g., the ratio ofL-amino acid entity to R-amino acid entity is about 2:3;

k) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is at least 1:4, or least 1:3, and not more than 3:4, e.g., theratio of the L-amino acid entity to the L-glutamine or a salt thereof isabout 1:2;

l) the ratio of the R-amino acid entity to the L-glutamine or a saltthereof is at least 1:4, or least 1:2, and not more than 6:7, e.g., theratio of the R-amino acid entity to the L-glutamine or a salt thereof isabout 3:4; or

m) a combination of two or three of (j)-(l).

508. The composition of any of the preceding embodiments, furthercomprising one or both of an isoleucine (I)-amino acid-entity and avaline (V)-amino acid-entity, e.g., both the I-amino acid-entity and theV-amino acid-entity are present.

509. The composition of embodiment 508, wherein:

n) the wt. % of the L-amino acid-entity in the composition is greaterthan or equal to the wt. % of the I-amino acid-entity and the V-aminoacid-entity in combination;

o) the wt. % of the L-amino acid-entity, the I-amino acid-entity, andthe V-amino acid-entity in combination in the composition is greaterthan or equal to the wt. % of the L-glutamine or a salt thereof;

p) the wt. % of the L-amino acid-entity, the I-amino acid-entity, andthe V-amino acid-entity in combination in the composition is less thanthe wt. % of the R-amino acid entity;

q) the wt. % of the R-amino acid entity and the L-glutamine or a saltthereof in the composition is greater than the wt. % of the L-aminoacid-entity, the I-amino acid-entity, and the V-amino acid-entity incombination; or

r) a combination of two, three, or four of (n)-(q).

510. The composition of embodiment 508 or 509, wherein:

s) the wt. % of the R-amino acid entity, the L-glutamine or a saltthereof, and the NAC or a salt thereof is at least 50% of thecomposition, or at least 70% of the composition, but not more than 90%of the composition;

t) the wt. % of the NAC or a salt thereof is at least 1%, or at least2%, but not more than 10% of the composition;

u) the wt. % of the L-amino acid-entity, the I-amino acid-entity, andthe V-amino acid-entity in combination is at least 15%, or at least 20%,but not more than 50% of the composition;

v) the wt. % of the R-amino acid entity, the L-glutamine or a saltthereof, and the NAC or a salt thereof is at least 40%, or at least 50%,but not more than 80% of the composition; or

w) a combination of two, three, or four of (s)-(v).

511. The composition of any of embodiments 508-510, wherein:

x) the ratio of the L-amino acid entity to the I-amino acid entity is atleast 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not morethan 3:1, e.g., the ratio of the L-amino acid entity to the I-amino acidentity is about 2:1;

y) the ratio of L-amino acid entity to V-amino acid entity is at least1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or not more than3:1, e.g., the ratio of L to V is about 2:1;

z) the ratio of the L-amino acid entity to the R-amino acid entity isgreater than 1:3, greater than 1.5 to 3, and less than 3:3, e.g., theratio of the L-amino acid entity to the R-amino acid entity is about2:3;

aa) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is greater than 1:4, greater than 1.5 to 4 and less than 4:4, orless than 3:4, e.g., the ratio of the L-amino acid entity to theL-glutamine or a salt thereof is about 1:2; or

bb) a combination of two, three, or four of (x)-(aa).

512. The composition of any of embodiments 508-511, wherein:

cc) the ratio of the I-amino acid entity to the V-amino acid entity isat least 0.5:1, or at least 0.75:1, and not more than 1.5 to 1 or notmore than 2:1, e.g., the ratio of the L-amino acid entity to the I-aminoacid entity is about 1:1;

dd) the ratio of the I-amino acid entity to the R-amino acid entity isat least 0.5:3, or at least 0.75:3, and not more than 2:3, or not morethan 1.5:3, e.g., the ratio of the L-amino acid entity to the I-aminoacid entity is about 1:3;

ee) the ratio of the I-amino acid entity to the L-glutamine or a saltthereof is at least 0.5:4, or at least 0.75:4, and not more than 3:4, ornot more than 2:4, e.g., the ratio of the L-amino acid entity to theL-glutamine or a salt thereof is about 1:4; or

ff) or a combination of two or three of (cc)-(ee).

513. The composition of any of embodiments 508-512, wherein:

gg) the ratio of the L-amino acid entity to the V-amino acid entity isat least 1.5:1, or at least 1.75:1, and not more than 2.5 to 1 or notmore than 3:1, e.g., is the ratio of the L-amino acid entity to theV-amino acid entity is about 2:1;

hh) the ratio of the L-amino acid entity to the R-amino acid entity isgreater than 1:3, greater than 1.5 to 3, and less than 3:3, e.g., theratio of the L-amino acid entity to the R-amino acid entity is about2:3;

ii) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is greater than 1:4, greater than 1.5 to 4 and less than 4:4, orless than 3:4, e.g., the ratio of the L-amino acid entity to theL-glutamine or a salt thereof is about 1:2; or

jj) a combination of two or three of (gg)-(ii).

514. The composition of any of embodiments 508-513, wherein:

kk) the ratio of the V-amino acid entity to the L-glutamine or a saltthereof is at least 0.5:4, or at least 0.75:4, and not more than 3:4, ornot more than 2:4, e.g., the ratio of the L-amino acid entity to theL-glutamine or a salt thereof is about 1:4;

ll) the ratio of the V-amino acid entity to the R-amino acid entity isat least 0.5:3, or at least 0.75:3, and not more than 2:3, or not morethan 1.5:3, e.g., the ratio of the V-amino acid entity to the R-aminoacid entity is about 1:3;

mm) the ratio of the L-amino acid-entity, the I-amino acid-entity, andthe V-amino acid-entity in combination to the R-amino acid entity,L-glutamine or a salt thereof, and NAC or a salt thereof is at least1:4, or at least 2:3, or not more than 5:7, or not more than 6:7, e.g.,the ratio is about 6:11; or

nn) a combination of two or three of (kk)-(mm).

515. The composition of any of the preceding embodiments, wherein:

oo) a wt. % of the L-amino acid entity in the composition is greaterthan the wt. % of the NAC or a salt thereof;

pp) a wt. % of the R-amino acid entity in the composition is greaterthan the wt. % of the NAC or a salt thereof;

qq) a wt. % of the L-glutamine or a salt thereof in the composition isgreater than the wt. % of the NAC or a salt thereof; or

rr) a combination of two or three of (oo)-(qq).

516. The composition of any of the preceding embodiments, wherein atleast one of the amino acids of (a)-(d) is a free amino acid, e.g., two,three, or four of the amino acids of (a)-(d) are a free amino acid,e.g., at least 50 wt. % of the total wt. of the composition is one ormore amino acid entities in free form.

517. The composition of any of the preceding embodiments, wherein atleast one of the amino acids of (a)-(d) is in a salt form, e.g., one,two, three, or four of the amino acids of (a)-(d) is in a salt form,e.g., at least 10 wt. % of the total wt. of the composition is one ormore amino acid entities in salt form.

518. The composition of embodiment 517, wherein at least 10 wt. % of thetotal wt. of the composition is one or more amino acid entities in saltform.

519. The composition of any of the preceding embodiments, wherein thecomposition is capable of one, two, three, four, five, or all of:

a) decreasing or preventing liver fibrosis;

b) decreasing or preventing liver injury;

c) decreasing or preventing hepatocyte inflammation;

d) improving, e.g., increasing, glucose tolerance;

e) decreasing or preventing steatosis;

f) decreasing or preventing hepatocyte ballooning; or

g) improving gut function.

520. The composition of any of the preceding embodiments, wherein thecomposition further comprises a serine (S)-amino acid entity, e.g., aS-amino acid entity chosen from L-serine, phosphoserine,P-hydroxypyruvate, L-glycine, tryptophan, acetylserine, cystathionine,cysteine, phosphatidylserine, and D-serine or a combination thereof,e.g., a combination of L-serine and L-glycine.

521. The composition of embodiment 520, wherein the compositioncomprises an L-amino acid entity, an I-amino acid entity, an V-aminoacid entity, an R-amino acid entity, an L-glutamine or a salt thereof,an NAC or a salt thereof, and an L-glycine.

522. The composition of embodiment 520, wherein the compositioncomprises an L-amino acid entity, an I-amino acid entity, an V-aminoacid entity, an R-amino acid entity, an L-glutamine or a salt thereof,an NAC or a salt thereof, and an L-serine.

523. The composition of embodiment 520, wherein the compositioncomprises an L-amino acid entity, an I-amino acid entity, an V-aminoacid entity, an R-amino acid entity, an L-glutamine or a salt thereof,an NAC or a salt thereof, an L-glycine, and an L-serine.

524. The composition of any of embodiments 520-523, wherein the wt.ratio of the L-amino acid entity, the I-amino acid entity, the V-aminoacid entity, the R-amino acid entity, the L-glutamine or salt thereof,and the NAC or salt thereof is about 1:0.5:0.5:1.5:2:0.15 or about1:0.5:0.5:1.5:2:0.3.

525. The composition of any of the preceding embodiments, wherein thewt. ratio of the L-amino acid entity, the R-amino acid entity, theL-glutamine or a salt thereof, and the NAC or salt thereof is about 0.5to 3:0.5 to 4:1 to 4:0.1 to 2.5, e.g., the wt. ratio of the L-amino acidentity, the R-amino acid entity, the L-glutamine or a salt thereof, andthe NAC or salt thereof is about 1:1.5:2:0.15 or about 1:1.5:2:0.3.

526. The composition of embodiment 525, wherein the wt. ratio of theL-amino acid entity, the R-amino acid entity, the L-glutamine or a saltthereof, and the NAC or salt thereof is about 1:0.75:2:0.15 or about1:0.75:2:0.3.

527. The composition of any of the preceding embodiments, wherein thewt. ratio of the L-amino acid entity, the I-amino acid entity, theV-amino acid entity, the R-amino acid entity, the L-glutamine or saltthereof, and the NAC or salt thereof is about 1:0.5:0.5:1.5:2:0.15 orabout 1:0.5:0.5:1.5:2:0.3.

528. The composition of any of embodiments 13-33, wherein thecomposition comprises about 0.5 g to about 10 g of the L-amino acidentity, about 0.25 g to about 5 g of the I-amino acid entity, about 0.25g to about 5 g of the V-amino acid entity, about 0.5 g to about 20 g ofthe R-amino acid entity, about 1 g to about 20 g of the L-glutamine or asalt thereof, and about 0.1 g to about 5 g of the NAC or salt thereof,e.g., the composition comprises about 1 g of the L-amino acid entity,about 0.5 g of the I-amino acid entity, about 0.5 g of V-amino acidentity, about 1.5 g of R-amino acid entity, about 2 g of L-glutamine ora salt thereof, and about 0.15 g or about 0.3 g of NAC or salt thereof.

529. The composition of embodiment 528, wherein the compositioncomprises about 4 g of the L-amino acid entity, about 2 g of the I-aminoacid entity, about 1 g of V-amino acid entity, about 3 g of R-amino acidentity, about 4 g of L-glutamine or a salt thereof, and about 0.9 g ofNAC or a salt thereof.

530. The composition of any of the preceding embodiments, wherein thecomposition comprises:

-   -   a) L-leucine or a salt thereof;    -   b) L-isoleucine or a salt thereof;    -   c) L-valine or a salt thereof;    -   b) L-arginine or a salt thereof;    -   e) L-glutamine or a salt thereof; and    -   f) NAC or a salt thereof.

531. The composition of embodiment 530, wherein the L-Leucine isprovided as part of a dipeptide comprising L-Leucine, or a salt thereof,or a tripeptide comprising L-Leucine, or a salt thereof.

532. The composition of embodiment 530 or 531, wherein the L-Isoleucineis provided as part of a dipeptide comprising L-Isoleucine, or a saltthereof, or a tripeptide comprising L-Isoleucine, or a salt thereof.

533. The composition of any of embodiments 530-532, wherein the L-Valineis provided as part of a dipeptide comprising L-Valine, or a saltthereof, or a tripeptide comprising L-Valine, or a salt thereof.

534. The composition of any of embodiments 530-533, wherein theL-Arginine is provided as part of a dipeptide comprising L-Arginine, ora salt thereof, or a tripeptide comprising L-Arginine, or a saltthereof.

535. The composition of any of embodiments 530-534, wherein theL-Glutamine is provided as part of a dipeptide comprising L-Glutamine,or a salt thereof, or a tripeptide comprising L-Glutamine, or a saltthereof.

536. The composition of any of embodiments 530-535, wherein the NAC isprovided as a part of a dipeptide comprising NAC, or a salt thereof, ora tripeptide comprising NAC, or a salt thereof.

537. The composition of any of the preceding embodiments, wherein thecomposition comprises a combination of 4 to 20 different amino acidentities, e.g., a combination of 5 to 15 different amino acid entities.

538. The composition of any of the preceding embodiments, wherein atleast two, three, four, or more amino acid entities is not a peptide ofmore than 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or20 amino acid residues in length.

539. A method for improving liver function, wherein the method comprisesadministering to a subject in need thereof an effective amount of acomposition of any of the preceding embodiments.

540. The method of embodiment 539, wherein the L-Leucine is provided aspart of a dipeptide comprising L-Leucine, or a salt thereof, or atripeptide comprising L-Leucine, or a salt thereof.

541. The method of embodiment 539 or 540, wherein the L-Arginine isprovided as part of a dipeptide comprising L-Arginine, or a saltthereof, or a tripeptide comprising L-Arginine, or a salt thereof.

542. The method of any of embodiments 539-541, wherein the L-Glutamineis provided as part of a dipeptide comprising L-Glutamine, or a saltthereof, or a tripeptide comprising L-Glutamine, or a salt thereof.

543. The method of any of embodiments 539-542, wherein the NAC isprovided as a part of a dipeptide comprising NAC, or a salt thereof, ora tripeptide comprising NAC, or a salt thereof.

544. A method for treating one or more symptoms selected from the groupconsisting of decreased fat metabolism, hepatocyte apoptosis, hepatocyteballooning, inflammation of adipose tissue, inflammation of hepatictissue, fibrosis, liver injury, steatosis, glucose tolerance, andoxidative stress, wherein the method comprises administering to asubject in need thereof an effective amount of a composition of any ofthe preceding embodiments.

545. The method of embodiment 544, wherein the L-Leucine is provided aspart of a dipeptide comprising L-Leucine, or a salt thereof, or atripeptide comprising L-Leucine, or a salt thereof.

546. The method of embodiment 544 or 545, wherein the L-Arginine isprovided as part of a dipeptide comprising L-Arginine, or a saltthereof, or a tripeptide comprising L-Arginine, or a salt thereof.

547. The method of any of embodiments 544-546, wherein the L-Glutamineis provided as part of a dipeptide comprising L-Glutamine, or a saltthereof, or a tripeptide comprising L-Glutamine, or a salt thereof.

548. The method of any of embodiments 544-547, wherein the NAC isprovided as a part of a dipeptide comprising NAC, or a salt thereof, ora tripeptide comprising NAC, or a salt thereof.

549. A method for treating fatty liver disease, wherein the methodcomprises administering to a subject in need thereof an effective amountof a compositionof any of the preceding embodiments.

550. The method of embodiment 549, wherein the L-Leucine is provided aspart of a dipeptide comprising L-Leucine, or a salt thereof, or atripeptide comprising L-Leucine, or a salt thereof.

551. The method of embodiment 549 or 550, wherein the L-Arginine isprovided as part of a dipeptide comprising L-Arginine, or a saltthereof, or a tripeptide comprising L-Arginine, or a salt thereof.

552. The method of any of embodiments 549-551, wherein the L-Glutamineis provided as part of a dipeptide comprising L-Glutamine, or a saltthereof, or a tripeptide comprising L-Glutamine, or a salt thereof.

553. The method of any of embodiments 549-552, wherein the NAC isprovided as a part of a dipeptide comprising NAC, or a salt thereof, ora tripeptide comprising NAC, or a salt thereof.

554. The method of any of the preceding embodiments, wherein the subjecthas a disease or disorder selected from the group consisting ofnon-alcoholic fatty liver (NAFL), non-alcoholic fatty liver disease(NAFLD), non-alcoholic steatohepatitis (NASH), alcoholic fatty liverdisease (AFLD), and alcoholic steatohepatitis (ASH).

555. The method of embodiment 554, wherein the subject has pediatricNAFLD.

556. The method of any of the preceding embodiments, wherein the subjecthas a high BMI, obesity, gut leakiness, gut dysbiosis or gut microbiomedisturbance.

557. The method of any of the preceding embodiments, wherein the subjecthas cirrhosis, hepatocarcinoma, an increased risk of liver failure, anincreased risk of death, metabolic syndrome, or type 2 diabetes.

558. The method of any of the preceding embodiments, wherein the subjecthas increased levels of inflammatory cytokines relative to a normalsubject, e.g., the subject has increased levels of TNFα relative to anormal subject e.g., without the one or more symptoms or without thefatty liver disease.

559. The method of any of the preceding embodiments, wherein the patientexhibits muscle atrophy or has a decreased ratio of muscle tissue toadipose tissue relative to a normal subject, e.g., without the one ormore symptoms or without a fatty liver disease, e.g., the patientexhibits muscle atrophy without one or both of fibrosis or cirrhosis.

560. The method of any of the preceding embodiments, wherein the subjectexhibits reverse lipid transport from adipose tissue to liver tissue.

561. The composition of any of the preceding embodiments, wherein thecomposition comprises free amino acids, wherein the amino acids comprisearginine, glutamine, N-acetylcysteine, and a branched-chain amino acidchosen from one, two, or all of leucine, isoleucine, and valine.

562. The composition of embodiment 561, wherein the branched-chain aminoacid is leucine, isoleucine, and valine.

563. The composition of embodiment 561 or 562, wherein the wt ratio ofleucine, isoleucine, valine, arginine, glutamine, N-acetylcysteine is1:0.5:0.5:1.5:2:0.15.

564. The composition of any of embodiments 561-563, wherein a totalweight (wt) of the amino acids is about 2 g to about 60 g.

565. The composition of embodiment 564, wherein the total wt of theamino acids is about 6 g, about 12 g, about 18 g, about 24 g, or about48 g.

566. The composition of any of embodiments 561-565, wherein thecomposition comprises about 0.5 g to about 10 g of leucine, about 0.25 gto about 5 g of isoleucine, about 0.25 g to about 5 g of valine, about 1g to about 20 g of arginine, about 1 g to about 20 g of glutamine, andabout 0.1 g to about 5 g of N-acetylcysteine.

567. The composition of embodiment 566, wherein the compositioncomprises about 1 g of leucine, about 0.5 g of isoleucine, about 0.5 gof valine, about 1.5 g of arginine, about 2 g of glutamine, and about0.15 g of N-acetylcysteine.

568. The composition of embodiment 566, wherein the compositioncomprises about 2 g of leucine, about 1 g of isoleucine, about 1 g ofvaline, about 3.0 g of arginine, about 4 g of glutamine, and about 0.3 gof N-acetylcysteine.

569. The composition of embodiment 566, wherein the compositioncomprises about 4 g of leucine, about 2 g of isoleucine, about 2 g ofvaline, about 6.0 g of arginine, about 8 g of glutamine, and about 0.6 gof N-acetylcysteine.

570. The composition of any of embodiments 561-566, wherein the aminoacids comprise about 10 wt % to about 30 wt % leucine, about 5 wt % toabout 15 wt % isoleucine, about 5 wt % to about 15 wt % valine, about 15wt % to about 40 wt % arginine, about 20 wt % to about 50 wt %glutamine, and about 1 wt % to about 8 wt % N-acetylcysteine.

571. The composition of embodiment 570, wherein the amino acids compriseabout 16 wt % to about 18 wt % leucine, about 7 wt % to about 9 wt %isoleucine, about 7 wt % to about 9 wt % valine, about 28 wt % to about32 wt % arginine, about 31 wt % to about 34 wt % glutamine, and about 1wt % to about 5 wt % N-acetylcysteine.

572. The composition of embodiment 571, wherein the amino acids compriseabout 16.8 wt % leucine, about 8.4 wt % isoleucine, about 8.4 wt %valine, about 30.4 wt % arginine, about 33.6 wt % glutamine, and about2.5 wt % N-acetylcysteine.

573. The composition of any of the preceding embodiments, wherein thecomposition further comprises one or more pharmaceutically acceptableexcipients.

574. The composition of embodiment 573, wherein the excipients areselected from the group consisting of citric acid, lecithin, asweetener, a dispersion enhancer, a flavoring, a bitterness maskingagent, and a natural or artificial coloring.

575. The composition of any of the preceding embodiments, wherein thecomposition is in the form of a solid, powder, solution, or gel.

576. The composition of any of the preceding embodiments, wherein theamino acids consist of leucine, isoleucine, valine, arginine, glutamineand N-acetylcysteine.

577. A method for treating one or more symptoms selected from the groupconsisting of decreased fat metabolism, hepatocyte apoptosis, hepatocyteballooning, inflammation of adipose tissue, inflammation of hepatictissue, fibrosis, and oxidative stress, wherein the method comprisesadministering to a subject in need thereof an effective amount of thecomposition of any one of embodiments 561-576.

578. The method of embodiment 577, wherein the subject has non-alcoholicfatty liver disease (NAFLD).

579. The method of embodiment 577 or 578, wherein the subject haspediatric NAFLD.

580. The method of embodiment 578 or 579, wherein the patient hassteatosis.

581. The method of embodiment 577, wherein the subject has non-alcoholicsteatohepatitis (NASH).

582. The method of embodiment 581, wherein the subject has fibrosis.

583. The method of embodiment 577, wherein the subject has cirrhosis.

584. The method of embodiment 583, wherein the subject hashepatocarcinoma, an increased risk of liver failure, or an increasedrisk of death.

585. The method of any of embodiments 577-584, wherein the subject hastype 2 diabetes.

586. A method for treating non-alcoholic fatty liver disease (NAFLD)comprising administering to a subject in need thereof an effectiveamount of the composition of any of embodiments 561-576.

587. The method of embodiment 586, wherein the subject has pediatricNAFLD.

588. The method of embodiment 586 or 587, wherein the patient hassteatosis.

589. A method for treating non-alcoholic steatohepatitis (NASH)comprising administering to a subject in need thereof an effectiveamount of the composition of any of embodiments 561-576.

590. The method of embodiment 589, wherein the subject has fibrosis.

591. A method for treating cirrhosis comprising administering to asubject in need thereof an effective amount of the composition of any ofembodiments 561-576.

592. The method of embodiment 591, wherein the subject hashepatocarcinoma, an increased risk of liver failure, or an increasedrisk of death.

593. The method of any of embodiments 577-592, wherein administering thecomposition results in an improvement in one or more metabolic symptomsin the subject.

594. The method of embodiment 593, wherein the improvement in one ormore metabolic symptoms is selected from the following: increased freefatty acid and lipid metabolism, improved mitochondrial function, whiteadipose tissue (WAT) browning, decreased reactive oxygen species (ROS),increased levels of glutathione (GSH), decreased hepatic inflammation,decreased hepatocyte ballooning, improved gut barrier function,increased insulin secretion, or glucose tolerance.

595. The method of embodiment 594, wherein the increased free fatty acidand lipid metabolism occurs in the liver.

596. The method of embodiment 594 or 595, wherein administration of thecomposition results in an improvement in one or more metabolic symptomsafter a treatment period of 24 hours.

597. The method of any of embodiments 577-596, wherein the methodfurther comprises determining the level of one, two, three, four, five,six, seven, eight, nine, ten, or more (e.g., all) of the following:

-   -   a) alanine aminotransferase (ALT);    -   b) aspartate aminotransferase (AST);    -   c) adiponectin;    -   d) N-terminal fragment of type III collagen (proC3);    -   e) caspase-cleaved keratin 18 fragments (M30 and M65);    -   f) IL-1 beta;

g) C-reactive protein;

h) PIIINP;

i) TIMP1;

j) MCP-1; or

k) FGF-21.

598. The method of embodiment 597, wherein administration of thecomposition results in an improvement in one or more of a)-k) after atreatment period of 24 hours.

599. The method of any of embodiments 577-598, wherein the compositionis administered prior to a meal.

600. The method of any of embodiments 577-598, wherein the compositionis administered concurrent with a meal.

601. The method of any of embodiments 577-598, wherein the compositionis administered following a meal.

602. The method of any of embodiments 577-601, wherein the compositionis administered with a second agent.

603. The method of embodiment 602, wherein the second agent is selectedfrom the group consisting of a farnesoid X receptor (FXR) agonist, astearoyl CoA desaturase inhibitor, a CCR2 and CCR5 chemokine antagonist,a PPAR alpha and delta agonist, a caspase inhibitor, a galectin-3inhibitor, an acetyl CoA carboxylase inhibitor, or an ileal sodium bileacid co-transporter inhibitor.

604. A dietary composition comprising the composition of any ofembodiments 561-576, e.g., wherein the dietary composition is chosenfrom a medical food, a functional food, or a supplement.

605. The composition of any of embodiments 561-576 for use as a dietarycomposition, e.g., wherein the dietary composition is chosen from amedical food, a functional food, or a supplement.

606. The dietary composition of embodiment 605, wherein the subject hastype 2 diabetes and/or a relatively high BMI.

607. The dietary composition of any of embodiments 605 or 606, whereinthe subject has non-alcoholic fatty liver disease (NAFLD).

608. The dietary composition of any of embodiments 605-607, wherein thesubject has pediatric NAFLD.

609. The dietary composition of any of embodiments 605-608, wherein thepatient has steatosis.

610. The dietary composition of any of embodiments 605-609, wherein thesubject has non-alcoholic steatohepatitis (NASH).

611. The dietary composition of embodiment 610, wherein the subject hasfibrosis.

612. The dietary composition of any of embodiments 604-606, wherein thesubject has cirrhosis.

613. The dietary composition of embodiment 612, wherein the subject hashepatocarcinoma, an increased risk of liver failure, or an increasedrisk of death.

614. The dietary composition of any of embodiments 604-613, wherein thesubject has type 2 diabetes.

615. The dietary composition of any of embodiments 604-614, wherein thecomposition promotes weight loss in the subject.

616. The method or dietary composition of any of the precedingembodiments, wherein the composition is administered at a dose of about15 g/d to about 90 g/d.

617. The method or dietary composition of embodiment 616, wherein thecomposition is administered at a dose of about 18 g/d, about 24 g/d,about 36/d, about 54 g/d, or about 72 g/d.

618. The method or dietary composition of any of the precedingembodiments, wherein the composition is administered one, two, to threetimes per day.

619. The method or dietary composition of any of the precedingembodiments, wherein the composition is administered at a dose of about6 g, about 8 g, about 12 g, about 16 g, about 18 g, or about 24 g threetimes per day.

620. The composition of any of the preceding embodiments, wherein:

1) the ratio of the L-amino acid entity to the I-amino acid entity is atleast 1.5:1, or at least 1.75:1, and not more than 2.5:1 or not morethan 3:1, e.g., the ratio of the L-amino acid entity to the I-amino acidentity is about 2:1;

2) the ratio of L-amino acid entity to V-amino acid entity is at least2:1, at least 3:1, at least 3.5:1, at least 4:1, or at least 5:1, andnot more than 6:1, e.g., the ratio of L-amino acid entity to V-aminoacid entity is about 4:1;

3) the ratio of the L-amino acid entity to the R-amino acid entity is atleast 1:1, at least 3.5:3, at least 4:3, or at least 2:1, and not morethan 5:2, e.g., the ratio of the L-amino acid entity to the R-amino acidentity is about 4:3;

4) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is at least 0.5:1, or at least 0.75:1, and not more than 1.5 to1 or not more than 2:1, e.g., the ratio of the L-amino acid entity tothe L-glutamine or salt thereof is about 1:1;

5) the ratio of the L-amino acid entity to the NAC entity or a saltthereof is at least 2:1, at least 3:1, at least 3.5:1, or at least 4:1,and not more than 5 to 1 or not more than 6:1, e.g., the ratio of theL-amino acid entity to the NAC entity or salt thereof is about 4:1(e.g., 4:0.9);

6) optionally wherein the ratio of the L-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:3, greater than1.5:3, about 2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g.,the ratio of the L-amino acid entity to the S-amino acid entity is about2:3, or the ratio of the L-amino acid entity to the S-amino acid entityis about 3:5; or

7) a combination of two, three, four, five, or six of (1)-(6).

621. The composition of embodiment 620, wherein:

8) the ratio of I-amino acid entity to V-amino acid entity is at least1:1, at least 1.5:1, or about 2:1, and not more than 2.5:1 or not morethan 3:1, e.g., the ratio of I-amino acid entity to V-amino acid entityis about 2:1;

9) the ratio of the I-amino acid entity to the R-amino acid entity isgreater than 1:3, greater than 1.5:3, or about 2:3, and not more than2.5:3 or not more than 1:1, e.g., the ratio of the I-amino acid entityto the R-amino acid entity is about 2:3;

10) the ratio of the I-amino acid entity to the L-glutamine or a saltthereof is at least 1:4, at least 1:3, or about 1:2, and not more than1:1 or not more than 2:1, e.g., the ratio of the I-amino acid entity tothe L-glutamine or salt thereof is about 1:2;

11) the ratio of the I-amino acid entity to the NAC entity or a saltthereof is at least 1:1, at least 1.5:1, or about 2:1, and not more than2.5:1 or not more than 3:1, e.g., the ratio of the I-amino acid entityto the NAC entity or salt thereof is about 2:1 (e.g., 2:0.9);

12) optionally wherein the ratio of the I-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:4, greater than1.5:4, about 1:3, or about 3:10, and not more than 1.5:3 or 2:3, e.g.,the ratio of the I-amino acid entity to the S-amino acid entity is about1:3, or the ratio of the I-amino acid entity to the S-amino acid entityis about 3:10; or

13) a combination of two, three, four, or five of (8)-(12).

622. The composition of embodiment 620 or 621, wherein:

14) the ratio of the V-amino acid entity to the R-amino acid entity isgreater than 1:4, greater than 1.5:4, or about 1:3, and not more than1:2 or not more than 1:1, e.g., the ratio of the V-amino acid entity tothe R-amino acid entity is about 1:3;

15) the ratio of the V-amino acid entity to the L-glutamine or a saltthereof is greater than 1:5, or greater than 1.5:5, about 1:4, and notmore than 1.5:4 or not more than 1:3, e.g., the ratio of the V-aminoacid entity to the L-glutamine or salt thereof is about 1:4;

16) the ratio of the V-amino acid entity to the NAC entity or a saltthereof is at least 1:2, at least 1.5:2, or about 1:1, and not more than1.5:1 or not more than 2:1, e.g., the ratio of the V-amino acid entityto the NAC entity or salt thereof is about 1:1 (e.g., 1:0.9);

17) optionally wherein the ratio of the V-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:8, greater than1:7, about 1:6, or about 3:20, and not more than 1.5:6 or 1:3, e.g., theratio of the V-amino acid entity to the S-amino acid entity is about1:6, or the ratio of the V-amino acid entity to the S-amino acid entityis about 3:20; or

18) a combination of two, three, or four of (14)-(17).

623. The composition of any of embodiments 620-622, wherein:

19) the ratio of the R-amino acid entity to the L-glutamine or a saltthereof is greater than 1:2, greater than 1.25:2, or about 3:4, and notmore than 3.5:4 or not more than 1:1, e.g., the ratio of the R-aminoacid entity to the L-glutamine or salt thereof is about 3:4;

20) the ratio of the R-amino acid entity to the NAC entity or a saltthereof is at least 4:1, at least 4:1.5, or about 3:1, and not more than3:1.5 or not more than 3:2, e.g., the ratio of the R-amino acid entityto the NAC entity or salt thereof is about 3:1 (e.g., 3:0.9);

21) optionally wherein the ratio of the R-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:4, greater than1:3, about 1:2, or about 9:20, and not more than 1.5:2 or 1:1, e.g., theratio of the R-amino acid entity to the S-amino acid entity is about1:2, or the ratio of the R-amino acid entity to the S-amino acid entityis about 9:20; or

22) a combination of two or three of (19)-(21).

624. The composition of any of embodiments 620-623, wherein:

23) the ratio of the L-glutamine to the NAC entity or a salt thereof isat least 5:1, at least 5:1.5, or about 4:1, and not more than 4:1.5 ornot more than 3:1, e.g., the ratio of the L-glutamine to the NAC entityor salt thereof is about 4:1 (e.g., 4:0.9);

24) optionally wherein the ratio of the L-glutamine to the S-amino acidentity or a salt thereof is greater than 1:3, greater than 1.25:3, about2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g., the ratio ofthe L-glutamine to the S-amino acid entity is about 2:3, or the ratio ofthe L-glutamine to the S-amino acid entity is about 3:5; or

25) a combination of (23) and (24).

625. The composition of any of embodiments 620-624, wherein:

26) the ratio of the NAC entity to the S-amino acid entity or a saltthereof is greater than 1:8, greater than 1:7, or about 1:6, and notmore than 1:5 or not more than 1.5:5, e.g., the ratio of the NAC entityto the S-amino acid entity is about 1:6 (e.g., 0.9:6 or 2.7:20).

626. The composition of any of embodiments 620-625, wherein thecomposition satisfies the properties of (1)-(7) defined above.

627. The composition of any of embodiments 620-626, wherein thecomposition satisfies the properties of at least 2, 3, 4, 5, 6, or 7 ofany of properties (1)-(26) defined above.

628. The composition of any of embodiments 620-627, wherein the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, and NAC or a salt thereof is 12:6:3:9:12:2.7.

629. The composition of any of embodiments 620-628, wherein the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, and the S-amino acid entity is12:6:3:9:12:2.7:18.

630. The composition of any of embodiments 620-629, wherein the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, and the S-amino acid entity is12:6:3:9:12:2.7:20.

631. The composition of any of embodiments 620-630, wherein the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, and NAC or a salt thereof is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%.

632. The composition of any of embodiments 620-631, wherein the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, and the S-amino acid entity is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:18+/−15%.

633. The composition of any of embodiments 620-632, wherein the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, the S-amino acid entity, and theL-glycine is 12:6:3:9:12:2.7:9:9. In certain embodiments, the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, the S-amino acid entity, and theL-glycine is 12:6:3:9:12:2.7:10:10.

634. The composition of any of embodiments 620-633, wherein the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, the S-amino acid entity, and theL-glycine is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:9+/−15%:9+/−15%. Incertain embodiments, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, theS-amino acid entity, and the L-glycine is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:10+/−15%:10+/−15%.

635. A pharmaceutical composition comprising the composition of any ofthe preceding embodiments.

636. The composition of any of the preceding embodiments, wherein theL-amino acid entity is chosen from the group consisting of L-leucine,β-hydroxy-β-methybutyrate (HMB), oxo-leucine, isovaleryl-CoA, D-leucine,and n-acetyl-leucine, or a combination thereof.

637. The composition of any of the preceding embodiments, wherein theR-amino acid entity is chosen from the group consisting of L-arginine,ornithine, argininosuccinate, citrulline, aspartate, glutamate,agmatine, creatine, D-arginine, and N-acetyl-arginine, or a combinationthereof.

638. The composition of any of the preceding embodiments, wherein theQ-amino acid entity is chosen from the group consisting of L-glutamine,glutamate, carbamoyl-P, glutamate, D-glutamine, and n-acetylglutamine,or a combination thereof.

639. The composition of any of the preceding embodiments, wherein theNAC-amino acid entity is chosen from the group consisting of NAC,serine, acetylserine, cystathionine, glutathione, homocysteine,methionine, D-cysteine, L-cysteine, cysteamine, and cystine, or acombination thereof.

640. The composition of any of the preceding embodiments, wherein theS-amino acid entity is chosen from the group consisting of L-serine,phosphoserine, P-hydroxypyruvate, L-glycine, tryptophan, acetylserine,cystathionine, and phosphatidylserine.

641. A dietary composition comprising the composition of any of thepreceding embodiments, wherein the dietary compositions is chosen from amedical food, a functional food, or a supplement.

642. A method of providing amino acid entities to a subject comprisingadministering to the subject an effective amount of the composition ofany of the preceding embodiments.

643. A method of manufacturing or making a composition comprisingforming a composition comprising the following:

a) a L-amino acid entity,

b) an R-amino acid entity,

c) a Q-amino acid entity;

d) a NAC entity, e.g., NAC; and optionally, e) an S-amino acid entity;

provided that:

f) at least one amino acid entity is not provided as a peptide of morethan 20 amino acid residues in length, wherein:

(i) the amino acid entity of (a) is selected from Table 2; and

(ii) one or both of the R-amino acid entity and the Q-amino acid entityare present at a higher amount (wt. %) than the L-amino acid entity.

644. The method of any of the preceding embodiments, wherein:

1) the ratio of the L-amino acid entity to the I-amino acid entity is atleast 1.5:1, or at least 1.75:1, and not more than 2.5:1 or not morethan 3:1, e.g., the ratio of the L-amino acid entity to the I-amino acidentity is about 2:1;

2) the ratio of L-amino acid entity to V-amino acid entity is at least2:1, at least 3:1, at least 3.5:1, at least 4:1, or at least 5:1, andnot more than 6:1, e.g., the ratio of L-amino acid entity to V-aminoacid entity is about 4:1;

3) the ratio of the L-amino acid entity to the R-amino acid entity is atleast 1:1, at least 3.5:3, at least 4:3, or at least 2:1, and not morethan 5:2, e.g., the ratio of the L-amino acid entity to the R-amino acidentity is about 4:3;

4) the ratio of the L-amino acid entity to the L-glutamine or a saltthereof is at least 0.5:1, or at least 0.75:1, and not more than 1.5 to1 or not more than 2:1, e.g., the ratio of the L-amino acid entity tothe L-glutamine or salt thereof is about 1:1;

5) the ratio of the L-amino acid entity to the NAC entity or a saltthereof is at least 2:1, at least 3:1, at least 3.5:1, or at least 4:1,and not more than 5 to 1 or not more than 6:1, e.g., the ratio of theL-amino acid entity to the NAC entity or salt thereof is about 4:1(e.g., 4:0.9);

6) optionally wherein the ratio of the L-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:3, greater than1.5:3, about 2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g.,the ratio of the L-amino acid entity to the S-amino acid entity is about2:3, or the ratio of the L-amino acid entity to the S-amino acid entityis about 3:5; or

7) a combination of two, three, four, five, or six of (1)-(6).

645. The method of embodiment 644, wherein:

8) the ratio of I-amino acid entity to V-amino acid entity is at least1:1, at least 1.5:1, or about 2:1, and not more than 2.5:1 or not morethan 3:1, e.g., the ratio of I-amino acid entity to V-amino acid entityis about 2:1;

9) the ratio of the I-amino acid entity to the R-amino acid entity isgreater than 1:3, greater than 1.5:3, or about 2:3, and not more than2.5:3 or not more than 1:1, e.g., the ratio of the I-amino acid entityto the R-amino acid entity is about 2:3;

10) the ratio of the I-amino acid entity to the L-glutamine or a saltthereof is at least 1:4, at least 1:3, or about 1:2, and not more than1:1 or not more than 2:1, e.g., the ratio of the I-amino acid entity tothe L-glutamine or salt thereof is about 1:2;

11) the ratio of the I-amino acid entity to the NAC entity or a saltthereof is at least 1:1, at least 1.5:1, or about 2:1, and not more than2.5:1 or not more than 3:1, e.g., the ratio of the I-amino acid entityto the NAC entity or salt thereof is about 2:1 (e.g., 2:0.9);

12) optionally wherein the ratio of the I-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:4, greater than1.5:4, about 1:3, or about 3:10, and not more than 1.5:3 or 2:3, e.g.,the ratio of the I-amino acid entity to the S-amino acid entity is about1:3, or the ratio of the I-amino acid entity to the S-amino acid entityis about 3:10; or

13) a combination of two, three, four, or five of (8)-(12).

646. The method of embodiment 644 or 645, wherein:

14) the ratio of the V-amino acid entity to the R-amino acid entity isgreater than 1:4, greater than 1.5:4, or about 1:3, and not more than1:2 or not more than 1:1, e.g., the ratio of the V-amino acid entity tothe R-amino acid entity is about 1:3;

15) the ratio of the V-amino acid entity to the L-glutamine or a saltthereof is greater than 1:5, or greater than 1.5:5, about 1:4, and notmore than 1.5:4 or not more than 1:3, e.g., the ratio of the V-aminoacid entity to the L-glutamine or salt thereof is about 1:4;

16) the ratio of the V-amino acid entity to the NAC entity or a saltthereof is at least 1:2, at least 1.5:2, or about 1:1, and not more than1.5:1 or not more than 2:1, e.g., the ratio of the V-amino acid entityto the NAC entity or salt thereof is about 1:1 (e.g., 1:0.9);

17) optionally wherein the ratio of the V-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:8, greater than1:7, about 1:6, or about 3:20, and not more than 1.5:6 or 1:3, e.g., theratio of the V-amino acid entity to the S-amino acid entity is about1:6, or the ratio of the V-amino acid entity to the S-amino acid entityis about 3:20; or

18) a combination of two, three, or four of (14)-(17).

647. The method of any of embodiments 644-646, wherein:

19) the ratio of the R-amino acid entity to the L-glutamine or a saltthereof is greater than 1:2, greater than 1.25:2, or about 3:4, and notmore than 3.5:4 or not more than 1:1, e.g., the ratio of the R-aminoacid entity to the L-glutamine or salt thereof is about 3:4;

20) the ratio of the R-amino acid entity to the NAC entity or a saltthereof is at least 4:1, at least 4:1.5, or about 3:1, and not more than3:1.5 or not more than 3:2, e.g., the ratio of the R-amino acid entityto the NAC entity or salt thereof is about 3:1 (e.g., 3:0.9);

21) optionally wherein the ratio of the R-amino acid entity to theS-amino acid entity or a salt thereof is greater than 1:4, greater than1:3, about 1:2, or about 9:20, and not more than 1.5:2 or 1:1, e.g., theratio of the R-amino acid entity to the S-amino acid entity is about1:2, or the ratio of the R-amino acid entity to the S-amino acid entityis about 9:20; or

22) a combination of two or three of (19)-(21).

648. The method of any of embodiments 644-647, wherein:

23) the ratio of the L-glutamine to the NAC entity or a salt thereof isat least 5:1, at least 5:1.5, or about 4:1, and not more than 4:1.5 ornot more than 3:1, e.g., the ratio of the L-glutamine to the NAC entityor salt thereof is about 4:1 (e.g., 4:0.9);

24) optionally wherein the ratio of the L-glutamine to the S-amino acidentity or a salt thereof is greater than 1:3, greater than 1.25:3, about2:3, or about 3:5, and not more than 2.5:3 or 1:1, e.g., the ratio ofthe L-glutamine to the S-amino acid entity is about 2:3, or the ratio ofthe L-glutamine to the S-amino acid entity is about 3:5; or

25) a combination of (23) and (24).

649. The method of any of embodiments 644-648, wherein:

26) the ratio of the NAC entity to the S-amino acid entity or a saltthereof is greater than 1:8, greater than 1:7, or about 1:6, and notmore than 1:5 or not more than 1.5:5, e.g., the ratio of the NAC entityto the S-amino acid entity is about 1:6 (e.g., 0.9:6 or 2.7:20).

650. The method of any of embodiments 644-649, wherein the compositionsatisfies the properties of (1)-(7) defined above.

651. The method of any of embodiments 644-650, wherein the compositionsatisfies the properties of at least 2, 3, 4, 5, 6, or 7 of any ofproperties (1)-(26) defined above.

652. The method of any of embodiments 644-651, wherein the ratio of theL-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, and NAC or a salt thereof is 12:6:3:9:12:2.7.

653. The method of any of embodiments 644-652, wherein the ratio of theL-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, and the S-amino acid entity is12:6:3:9:12:2.7:18.

654. The method of any of embodiments 644-653, wherein the ratio of theL-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, and the S-amino acid entity is12:6:3:9:12:2.7:20.

655. The method of any of embodiments 644-654, wherein the ratio of theL-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, and NAC or a salt thereof is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%.

656. The method of any of embodiments 644-655, wherein the ratio of theL-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, and the S-amino acid entity is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:18+/−15%.

657. The method of any of embodiments 644-656, wherein the ratio of theL-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, the S-amino acid entity, and theL-glycine is 12:6:3:9:12:2.7:9:9. In certain embodiments, the ratio ofthe L-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, the S-amino acid entity, and theL-glycine is 12:6:3:9:12:2.7:10:10.

658. The method of any of embodiments 644-657, wherein the ratio of theL-amino acid-entity, the I-amino acid-entity, and the V-aminoacid-entity in combination to the R-amino acid entity, L-glutamine or asalt thereof, NAC or a salt thereof, the S-amino acid entity, and theL-glycine is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:9+/−15%:9+/−15%. Incertain embodiments, the ratio of the L-amino acid-entity, the I-aminoacid-entity, and the V-amino acid-entity in combination to the R-aminoacid entity, L-glutamine or a salt thereof, NAC or a salt thereof, theS-amino acid entity, and the L-glycine is12+/−15%:6+/−15%:3+/−15%:9+/−15%:12+/−15%:2.7+/−15%:10+/−15%:10+/−15%.

659. The composition or method of any of the preceding embodiments,wherein the composition is capable of enhancing fatty acid oxidation,e.g., one or both of reducing levels of unsaturated fatty acids orincreasing levels of acylcarnitine (e.g., in a STAM mouse model or aFATZO mouse model). In certain embodiments, the reduction in levels ofunsaturated fatty acids is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the level of changeshown in Table 53, e.g., measured as described in Example 9. In certainembodiments, the increase in levels of acylcarnitine is at least 25%,30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or99% of the level of change shown in Table 53, e.g., measured asdescribed in Example 9.

660. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, liverfibrosis or liver injury by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay ofalanine transaminase (ALT), e.g., an antibody-based detection assay,e.g., an ELISA, e.g., as described in Example 4, e.g., relative to areference composition (e.g., a vehicle control).

661. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, liverfibrosis or liver injury by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay ofaspartate transaminase (AST), e.g., an antibody-based detection assay,e.g., an ELISA, e.g., as described in Example 4, e.g., relative to areference composition (e.g., a vehicle control).

662. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, alaninetransaminase (ALT) by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of ALT,e.g., an antibody-based detection assay, e.g., an ELISA, e.g., asdescribed in Example 4, e.g., relative to a reference composition (e.g.,a vehicle control).

663. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, aspartatetransaminase (AST) by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay of AST,e.g., an antibody-based detection assay, e.g., an ELISA, e.g., asdescribed in Example 4, e.g., relative to a reference composition (e.g.,a vehicle control).

664. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, liverfibrosis or liver injury by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%,24%, 25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay ofhydroxyproline, e.g., an antibody-based detection assay, e.g., an ELISA,e.g., as described in Example 4, e.g., relative to a referencecomposition (e.g., a vehicle control).

665. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces,hydroxyproline levels by at least 5%, 10%, 15%, 20%, 21%, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, or 30%, as detected using an assay ofhydroxyproline, e.g., an antibody-based detection assay, e.g., an ELISA,e.g., as described in Example 4, e.g., relative to a referencecomposition (e.g., a vehicle control).

666. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, liverfibrosis or liver injury by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected usingLX-2 cells, e.g., levels of Col1a1, Acta2, and/or TIMP2 in LX-2 cells,e.g., as assessed using a nucleic acid amplification method, e.g., PCRor qRT-PCR, e.g., as described in Example 7, e.g., relative to areference composition (e.g., a vehicle control; an amino acidcomposition comprising L-leucine, L-isoleucine, L-valine; NAC; or anamino acid composition comprising L-arginine, L-glutamine, and NAC).

667. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, expressionof one or more collagen biomarkers (e.g., Col1a1, Acta2, and/or TIMP2)by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%,70%, 75%, 80%, 85%, or 90% as detected using LX-2 cells, e.g., levels ofCol1a1, Acta2, and/or TIMP2 in LX-2 cells, e.g., as assessed using anucleic acid amplification method, e.g., PCR or qRT-PCR, e.g., asdescribed in Example 7, e.g., relative to a reference composition (e.g.,a vehicle control; an amino acid composition comprising L-leucine,L-isoleucine, L-valine; an amino acid composition comprising L-arginine,L-glutamine, and NAC; or NAC).

668. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, expressionof one or more collagen biomarkers (e.g., Col1a1) by at least 5%, 10%,15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or90% as detected using primary hepatic stellate cells, e.g., levels ofCol1a1 in primary hepatic stellate cells, e.g., as assessed using anantibody-based detection assay, e.g., an ELISA, e.g., as described inExample 12, e.g., relative to a reference composition (e.g., a vehiclecontrol; an amino acid composition comprising L-leucine, L-isoleucine,L-valine; an amino acid composition comprising L-arginine, L-glutamine,and NAC; an amino acid composition comprising L-leucine, L-isoleucine,L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine;isoleucine; leucine; or NAC).

669. The composition or method of any of the preceding embodiments,wherein the composition is capable of increasing, or increases,expression of one or more collagen biomarkers (e.g., procollagen lal) byat least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,75%, 80%, 85%, or 90% as detected using primary hepatic stellate cells,e.g., levels of procollagen lalin primary hepatic stellate cells, e.g.,as assessed using an antibody-based detection assay, e.g., an ELISA,e.g., as described in Example 12, e.g., relative to a referencecomposition (e.g., a vehicle control; an amino acid compositioncomprising L-leucine, L-isoleucine, L-valine; an amino acid compositioncomprising L-arginine, L-glutamine, and NAC; an amino acid compositioncomprising L-leucine, L-isoleucine, L-valine, L-arginine, andL-glutamine; valine; glutamine; arginine; isoleucine; leucine; or NAC).

670. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, hepatocyteinflammation by at least 5%, 10%, 15%, 20%, 20%, 30%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% as detected using HepG2 cells,e.g., decreased activity, e.g., decreased TNFα-induced activity of NF-kBin a reporter assay in HepG2 cells, as described in Example 8, e.g.,relative to a reference composition (e.g., a vehicle control; an aminoacid composition comprising L-leucine, L-isoleucine, L-valine; an aminoacid composition comprising L-arginine, L-glutamine, and NAC; an aminoacid composition comprising L-leucine, L-isoleucine, L-valine,L-arginine, and L-glutamine; or NAC).

671. The composition or method of any of the preceding embodiments,wherein the composition is capable of reducing, or reduces, TNFα-inducedactivity of NF-kB in HepG2 cells by at least 5%, 10%, 15%, 20%, 20%,30%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% asdetected using HepG2 cells, e.g., decreased activity, e.g., decreasedTNFα-induced activity of NF-kB in a reporter assay in HepG2 cells, asdescribed in Example 8, e.g., relative to a reference composition (e.g.,a vehicle control; an amino acid composition comprising L-leucine,L-isoleucine, L-valine; an amino acid composition comprising L-arginine,L-glutamine, and NAC; an amino acid composition comprising L-leucine,L-isoleucine, L-valine, L-arginine, and L-glutamine; or NAC).

672. The composition or method of any of the preceding embodiments,wherein the composition is capable of increasing, or increases, glucosetolerance, e.g., in a STAM mouse model or in a FATZO mouse model, by atleast 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, as detected using anassay of glucose levels, e.g., using glucose oxidase, e.g., using aglucometer, e.g., as described in Example 5, e.g., relative to areference composition (e.g., a vehicle control or a positive control,e.g., metformin).

673. The composition or method of any of the preceding embodiments,wherein the composition is capable of increasing, or increases, bloodglucose metabolism, e.g., in a STAM mouse model or in a FATZO mousemodel, by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, asdetected using an assay of glucose levels, e.g., using glucose oxidase,e.g., using a glucometer, e.g., as described in Example 5, e.g.,relative to a reference composition (e.g., a vehicle control or apositive control, e.g., metformin).

674. The composition or method of any of the preceding embodiments,wherein the composition is capable of decreasing, or decreases,steatosis and/or inflammation by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%,8%, 9%, or 10%, as detected using an assay of MCP1/CCL2, e.g., inprimary hepatocytes, e.g., using an antibody-based detection assay,e.g., an ELISA, e.g., as described in Example 10, e.g., relative to areference composition (e.g., a vehicle control; an amino acidcomposition comprising L-leucine, L-isoleucine, L-valine; an amino acidcomposition comprising L-arginine, L-glutamine, and NAC; an amino acidcomposition comprising L-leucine, L-isoleucine, L-valine, L-arginine,and L-glutamine; valine; glutamine; arginine; isoleucine; leucine; orNAC).

675. The composition or method of any of the preceding embodiments,wherein the composition is capable of decreasing, or decreases,MCP1/CCL2 levels by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%,as detected using an assay of MCP1/CCL2, e.g., in primary hepatocytes,e.g., using an antibody-based detection assay, e.g., an ELISA, e.g., asdescribed in Example 10, e.g., relative to a reference composition(e.g., a vehicle control; an amino acid composition comprisingL-leucine, L-isoleucine, L-valine; an amino acid composition comprisingL-arginine, L-glutamine, and NAC; an amino acid composition comprisingL-leucine, L-isoleucine, L-valine, L-arginine, and L-glutamine; valine;glutamine; arginine; isoleucine; leucine; or NAC).

676. The composition or method of any of the preceding embodiments,wherein the composition is capable of decreasing, or decreases, TNFαinflammatory response by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or10%, as detected using an assay of MCP1/CCL2 or an assay of IL-6, e.g.,in primary hepatic stellate cells, e.g., using an antibody-baseddetection assay, e.g., an ELISA, e.g., as described in Example 11, e.g.,relative to a reference composition (e.g., a vehicle control; an aminoacid composition comprising L-leucine, L-isoleucine, L-valine; an aminoacid composition comprising L-arginine, L-glutamine, and NAC; an aminoacid composition comprising L-leucine, L-isoleucine, L-valine,L-arginine, and L-glutamine; valine; glutamine; arginine; isoleucine;leucine; or NAC).

677. The composition or method of any of the preceding embodiments,wherein the composition is capable of decreasing, or decreases,MCP1/CCL2 levels and/or IL-6 levels by at least 1%, 2%, 3%, 4%, 5%, 6%,7%, 8%, 9%, or 10%, as detected using an assay of MCP1/CCL2 or an assayof IL-6, e.g., in primary hepatic stellate cells, e.g., using anantibody-based detection assay, e.g., an ELISA, e.g., as described inExample 11, e.g., relative to a reference composition (e.g., a vehiclecontrol; an amino acid composition comprising L-leucine, L-isoleucine,L-valine; an amino acid composition comprising L-arginine, L-glutamine,and NAC; an amino acid composition comprising L-leucine, L-isoleucine,L-valine, L-arginine, and L-glutamine; valine; glutamine; arginine;isoleucine; leucine; or NAC).

678. The composition of any of the preceding embodiments for use as amedicament.

679. The composition of any of the preceding embodiments for use in amethod as disclosed herein.

680. The use of a composition of any of the preceding embodiments in themanufacture of a medicament.

681. The use of a composition of any of the preceding embodiments in themanufacture of a medicament for treating any of the disorders orconditions disclosed herein.

Although many of the above embodiments are shown in dependent form, itis contemplated that any of the embodiments or combinations thereof maybe in independent form.

EXAMPLES

The Example below is set forth to aid in the understanding of theinventions, but is not intended to, and should not be construed to,limit its scope in any way.

Example 1 Method of Producing the Amino Acid Compositions

The amino acid compositions of the instant disclosure and formulationsthereof may be made according to methods known in the art. They may alsobe made by the methods described below.

The starting materials (individual amino acids and excipients) areblended and sieved to generate a powder blend, which is filled intostick packs. The contents of the stick packs are dispersed in water attime of use for oral administration. An example of the mixing andreconstitution protocols, and stick pack formulations made thereby, areprovided below.

Mixing Protocol

1. Ingredients were weighed into a container.

-   -   2. The container was sealed and placed in a Turbula mixer and        contents mixed on low setting for 2 minutes.    -   3. The blended powder was sieved using a No. 14 screen and any        clumps not passing through the sieve were broken apart.    -   4. The blended and sieved powder was transferred back to the        container and mixed in a Turbula mixer on low for 10 minutes.

TABLE 13 Stick pack formulations Formulation of Formulation of AminoAcid Amino Acid Placebo Composition A-1 Composition A-2 IngredientAmount per stick pack (g) FUSI-BCAA ™ 0.00 2.00 2.00 Instantized Blend(2:1:1 L:I:V) (contains L-Leucine) N/A (1.00) (1.00) (containsL-Isoleucine) N/A (0.50) (0.50) (contains L-Valine) N/A (0.50) (0.50)L-Arginine HCl 0.00 1.50 1.81 L-Glutamine 0.00 2.00 2.00N-Acetylcysteine 0.00 0.25 0.15 Citric Acid 0.98 0.67 0.67 Lecithin(Alcolec 0.59 0.83 0.83 F100) Acesulfame Potassium 0.04 0.05 0.05Sucralose micronized 0.02 0.03 0.03 NF Xanthan Gum 0.24 0.24 0.24(Ticaxan Rapid-3) Vanilla Custard #4306 0.06 0.06 0.06 Maltrin QD M5005.75 0.00 0.00 maltodextrin NF Nat Orange WONF 0.36 0.36 0.36 #1326 Lime865.0032U 0.05 0.05 0.05 Lemon 862.2169U 0.05 0.05 0.05 BitternessMasking 0.12 0.12 0.12 936.2160U FD&C Yellow 6 0.01 0.01 0.01 FD&C Red40 (1:100 0.0667 0.00 0.0000 in M500) Total (g) 8.33 8.22 8.42

Reconstitution Protocol

Stick pack formulations were reconstituted according to the followingprotocol:

-   -   1. A total (g) “amount per stick pack” of powder blend was        weighed.        -   2. About 118.3 g (4 oz) of cold filtered water was weighed            into a sealable container.        -   3. The “amount per stick pack” of the powder blend was            transferred to the sealable container and the container was            sealed.        -   4. The container was shaken vigorously for 20 to 30 seconds.

Example 2 Analytical Characterization of the Amino Acid Compositions

Described below are methods used to characterize some of thephysicochemical properties in formulations of the amino acidcompositions prepared according to Example 1.

Identification and Assay. The identification and assay of % label claimof each amino acid present in Formulations of Amino Acid Composition A-1and Amino Acid Composition A-2 was evaluated by reversed phase HPLC.

Amino Acid Analysis

Briefly, an amino acid analysis method using reversed-phase highpressure liquid chromatography (HPLC) was developed to measure freeamino acid content (except for N-acetylcysteine) in formulations ofamino acid compositions described herein following resuspension. Columnand chromatographic conditions were modified from Agilent TechnicalNote: “Automated Amino Acid Analysis Using an Agilent Poroshell HPH-C18Column (Agilent Application Note 5991-5571EN)”. Primary amino acids inthe sample are derivatized online using the Agilent 1260 or 1290 UPLCwell-plate autosampler using o-phthaldialdehyde (OPA). Separation isachieved using an Agilent ZORBAX Eclipse Plus column (4.6 mm ID×100 mm,3.5 μm). The OPA-derivatives of primary amino acids are detected usingfluorescence (FLD) at 340 nm emission/450 nm excitation wavelengths andUV detection at 338 nm. Individual amino acids are expected to eluteaccording to known representative chromatograms of amino acid standards.Concentrations of amino acids in samples are determined by fitting asample peak area to a standard curve. Alternatively, amino acid analysismay be performed using derivatization with AccQ-Tag chemistry andstandards (Waters).

Amino Acid Analysis: N-Acetylcysteine

For N-acetylcysteine (NAC), an HPLC test method was developed based onthe United States Pharmacopeia Monograph Chapter 39 (USP <39>) for“Acetylcysteine” to determine the content of N-acetylcysteine ofreconstituted powder of formulations described herein. This HPLC methodinvolves the use of a reversed-phase column without any derivatizationstep. The separation was done using a column of C-18 backbone as thestationary phase, and 0.05 M KH₂PO₄ as the mobile phase. UV detectionwas performed at 214 nm. The column was then flushed with 5%acetonitrile to remove any residual sample components at the end of eachinjection. At the end of the sequence, a low flow “system flush”procedure involving stronger organic solvents is used to preserve thecolumn for storage. N-Acetylcysteine is expected to elute according toknown representative chromatograms of standards. Concentrations ofN-acetylcysteine in samples are determined by fitting a sample peak areato a standard curve.

Results for Formulation of Amino Acid Composition A-1 (compared totheoretical g per serving) are shown in Table 14. For glutamine, a meanmass per serving of 1.84 g was observed; compared to 2.00 g theoreticalper serving, this gives a % agreement (or % label claim value) of 92%.For arginine, a mean mass per serving of 1.69 g was observed; comparedto 1.50 g theoretical per serving, this gives a % agreement (or % labelclaim value) of 113%. For valine, a mean mass per serving of 0.51 g wasobserved; compared to 0.50 g theoretical per serving, this gives a %agreement (or % label claim value) of 101%. For isoleucine, a mean massper serving of 0.52 g was observed; compared to 0.50 g theoretical perserving, this gives a % agreement (or % label claim value) of 104%. Forleucine, a mean mass per serving of 1.04 g was observed; compared to1.00 g theoretical per serving, this gives a % agreement (or % labelclaim value) of 104%. For N-acetylcysteine (NAC), a mean mass perserving of 0.28 g was observed; compared to 0.25 g theoretical perserving, this gives a % agreement (or % label claim value) of 111%.Overall, the amino acids and amino acid derivatives in the Formulationof Amino Acid

Composition A-1 had a range of % label claims of 92-113%.

TABLE 14 % Label Claim Results: Formulation of Amino Acid CompositionA-1 g of Individual Amino Acid/per serving of Amino Acid Composition A-1Sample Name GLN ARG VAL ILE LEU NAC AA Comp A-1 1.84 1.70 0.51 0.52 1.050.28 AA Comp A-1 1.82 1.68 0.50 0.52 1.04 0.28 AA Comp A-1 1.84 1.700.51 0.52 1.05 0.28 Mean 1.84 1.69 0.51 0.52 1.04 0.28 Theoretical 2.001.50 0.50 0.50 1.00 0.25 g/serving for each AA % Agreement 92 113 101104 104 111 Observed/ Theoretical*100

Results for Formulation of Amino Acid Composition A-2 (compared totheoretical g per serving) are shown in Table 15. For glutamine, a meanmass per serving of 2.102 g was observed; compared to 2.00 g theoreticalper serving, this gives a % agreement (or % label claim value) of105.1%. For arginine, a mean mass per serving of 1.922 g was observed;compared to 1.5 g theoretical per serving, this gives a % agreement (or% label claim value) of 107.5%. For valine, a mean mass per serving of0.536 g was observed; compared to 0.50 g theoretical per serving, thisgives a % agreement (or % label claim value) of 107.5%. For isoleucine,a mean mass per serving of 0.531 g was observed; compared to 0.50 gtheoretical per serving, this gives a % agreement (or % label claimvalue) of 106.2%. For leucine, a mean mass per serving of 1.058 g wasobserved; compared to 1.00 g theoretical per serving, this gives a %agreement (or % label claim value) of 105.8%. For N-acetylcysteine(NAC), a mean mass per serving of 0.153 g was observed; compared to 0.15g theoretical per serving, this gives a % agreement (or % label claimvalue) of 101.7%. Overall, the amino acids and amino acid derivatives inthe Formulation of Amino Acid Composition A-2 had a range of mean %label claims of 101-107%. Individual samples had a range of % labelclaims of 98.3-108.8%.

TABLE 15 % Label Claim Results: Formulation of Amino Acid CompositionA-2 g of Individual Amino Acid/per serving of Amino Acid Composition A-2Sample Name GLN ARG VAL ILE LEU NAC AA Comp A-2 2.09 1.92 0.54 0.53 1.050.16 AA Comp A-2 2.10 1.90 0.53 0.53 1.06 0.15 AA Comp A-2 2.11 1.960.54 0.53 1.06 0.15 AA Comp A-2 2.11 1.90 0.54 0.53 1.06 0.15 Mean 2.1021.922 0.536 0.531 1.058 0.153 Theoretical 2.00 1.50 0.50 0.50 1.00 0.15g/serving for each AA % Agreement 105.1 106.6 107.5 106.2 105.8 101.7Observed/ Theoretical*100

Example 3 Pharmacokinetic Characterization of the Amino AcidCompositions

The amino acid compositions of the present disclosure were characterizedin rodent and human subjects for their pharmacokinetic effects on aminoacid concentrations in response to ingestion of the compositions.

Rat Pharmacokinetics

The pharmacokinetic effects of a formulation of Amino Acid CompositionA-1 were tested in rats. After an overnight fast, rats were given theformulation by oral gavage. Portal vein and jugular vein blood wascollected just before the dose, and at 5, 15, 30, 60, 120, 240 and 360minutes thereafter. Plasma concentration of amino acid levels weremeasured, and maximum concentration (C_(max)), time of maximumconcentration (T_(max)), and half-life (T_(1/2)) were determined.Maximum concentration values are corrected for baseline endogenous aminoacid levels. Results for rat PK studies are shown below in Tables 16-21.

TABLE 16 Leucine Rat PK - Formulation of Amino Acid Composition A-1 DoseT_(max) T_(1/2) (mg/kg) C_(max) (μM) (h) (h) 266 421 1.0 1.2

TABLE 17 Isoleucine Rat PK - Formulation of Amino Acid Composition A-1Dose T_(max) T_(1/2) (mg/kg) C_(max) (μM) (h) (h) 133 176 0.6 1.0

TABLE 18 Valine Rat PK - Formulation of Amino Acid Composition A-1 DoseT_(max) T_(1/2) (mg/kg) C_(max) (μM) (h) (h) 133 323 0.9 1.5

TABLE 19 Arginine Rat PK - Formulation of Amino Acid Composition A-1Dose T_(max) T_(1/2) (mg/kg) C_(max) (μM) (h) (h) 399 896 1.0 1.1

TABLE 20 Glutamine Rat PK - Formulation of Amino Acid Composition A-1Dose T_(max) T_(1/2) (mg/kg) C_(max) (μM) (h) (h) 531 300 1.8 4.1

TABLE 21 N-acetylcysteine Rat PK - Formulation of Amino Acid CompositionA-1 Dose T_(max) T_(1/2) (mg/kg) C_(max) (μM) (h) (h) 66 34 0.9 0.8

Allometric scaling was assumed to convert rat mg/kg doses into humanequivalent mg/kg doses. A comparison of these equivalent mg/kg doses andhuman gram doses gram (assuming bodyweight of 70 kg) is shown in Table22.

TABLE 22 Amino Acid Doses: Comparison of Rat and Human Dose Leu Ile ValArg Gln NAC Rat (mg/kg) 266 133 133 399 531 66 Human (mg/kg) 43 21 21 6486 11 Human (g) 3 1.5 1.5 4.5 6 0.75

Human Pharmacokinetics

The impacts of orally administered Formulation of Amino Acid CompositionA-1 prepared according to Example 1 on amino acid pharmacokinetics wasevaluated in six apparently healthy human subjects between the ages of18 and 40. Changes in plasma concentrations of amino acids in responseto ingestion of the Formulation of Amino Acid Composition A-1 at twodoses (High: 3 stick packs, ˜18 g of amino acids; vs. Low: 1 stick pack,˜6 g of amino acids) were determined. Blood samples (3 mL) werecollected after an initial baseline and in specific intervals thereafter[i.e., 0 (pre-administration), 15, 30, 60, 90, 120, 150, 180, 210, and240 minutes]. Plasma concentration of amino acid levels were measured,and maximum concentration (C_(max)), time of maximum concentration(T_(max)), half-life (T_(1/2)) and total exposure (area under curve forplasma concentration time-courses of leucine, isoleucine, valine,arginine and glutamine) were determined. Maximum concentration and totalexposure values are corrected for baseline endogenous amino acid levels.These results are shown in Table 23-27.

TABLE 23 Leucine Human PK - Formulation of Amino Acid Composition A-1Dose C_(max) T_(max) T_(1/2) Dose (g) (μM) (h) (h) AUC_(last) (μM-h)HIGH 3.0 294 0.8 1.1 471 LOW 1.0 117 0.8 1.3 153

TABLE 24 Isoleucine Human PK - Formulation of Amino Acid Composition A-1Dose C_(max) T_(max) T_(1/2) Dose (g) (μM) (h) (h) AUC_(last) (μM-h)HIGH 1.5 141 0.7 0.8 194 LOW 0.5 52 0.8 0.6 54

TABLE 25 Valine Human PK - Formulation of Amino Acid Composition A-1Dose C_(max) T_(max) T_(1/2) Dose (g) (μM) (h) (h) AUC_(last) (μM-h)HIGH 1.5 238 0.8 1.3 400 LOW 0.5 89 0.8 1.7 101

TABLE 26 Arginine Human PK - Formulation of Amino Acid Composition A-1Dose T_(max) T_(1/2) Dose (g) C_(max) (μM) (h) (h) AUC_(last) (μM-h)HIGH 4.5 177 0.8 1.6 311 LOW 1.5 69 0.8 1.3 111

TABLE 27 Glutamine Human PK - Formulation of Amino Acid Composition A-1Dose T_(max) T_(1/2) Dose (g) C_(max) (μM) (h) (h) AUC_(last) (μM-h)HIGH 6.0 190 0.9 2.9 332 LOW 2.0 103 1.1 3.0 186

Example 4 Therapeutic Amino Acid Composition A-1 Treatment ImprovesLiver Fibrosis in an Animal Model of Chemically Induced Fibrosis

Amino Acid Composition A-1 was tested for its ability to affect liverfibrosis in a model of chemically induced liver fibrosis. A commonlyused model of experimental hepatic fibrosis is induced chemically inmice using carbon tetrachloride; CCl₄ (Gideon Smith, Animal Models ofCutaneous and Hepatic Fibrosis; Progress in Molecular Biology andTranslational Science, Vol. 105, pp. 371-408). CCl₄ causes inflammation,hepatocyte damage, necrosis and fibrosis after 4 weeks of treatment andcirrhosis after 8 weeks. Liver fibrosis induced in mice by carbontetrachloride (CCl₄) resembles important properties of human liverfibrosis including inflammation, regeneration and fiber formation.

Animals

Male BALB/c mice 7 to 8 weeks of age were used for this study. Animalswere housed four per cage, kept on a standard 12 hr light cycle andgiven free access to water and standard mouse chow. Food and water wereavailable ad libitum.

Procedure

Animals were dosed with 5% CCl₄ or vehicle intraperitoneally (IP)typically 3 days a week for 4 weeks. CCl₄ was formulated weekly. 10ml/kg of Amino Acid Composition A-1 at 23 mg/ml, 76 mg/ml or 153 mg/mlwas dosed by oral gavage twice daily. Animals were weighed twice weeklyand blood was collected via retro-orbital sinus once per week for serum.After four weeks, blood was collected for serum isolation and mice wereeuthanized via cervical dislocation. Two lobes of liver were removed—theleft lobe was placed in a tube containing 10% formalin forhistopathology, while the right lobe was weighed and placed in abeadbeater tube containing 2.3 mm zirconia beads and 2× volume of 1:100protease inhibitor (Sigma Aldrich, #P8340). Tissue samples werehomogenized for 2 minutes in a beadbeater machine and immediately spundown at 3,000 rpm for 15 minutes at 4° C. Serum was analyzed for ALT/ASTlevels at weeks 2 and 4. Homogenized liver samples were furtherevaluated for Hydroxyproline (Hyp) content to identify formation ofliver fibrosis.

Hydroxyproline (week 4)

Hydroxyproline (4-hydroxyproline, Hyp) is a common nonproteinogenicamino acid and is used as an indirect measure of the amount of collagenpresent, indicative of fibrosis. Hepatic Hyp content levels inCCl₄-treated animals were significantly higher than vehicle treatedanimals. Data are mean±standard deviation (stdev); “Comp A-1”: AminoAcid Composition A-1; *p<0.05 compared to vehicle control by unpaired Ttest. Raw data are shown in Table 28.

TABLE 28 Hepatic Hyp content level results Hydroxyproline Vehicle/Vehicle/ Comp A-1, Comp A-1, Comp A-1, Sham CCL4 23 mg/ml 76 mg/ml 153mg/ml mean 0.160 0.263* 0.280 0.228 0.201 stdev 0.067 0.107 0.104 0.1240.057

AST Levels and ALT Levels

Aspartate transaminase (AST) and alanine transaminase (ALT) are commonlymeasured clinical biomarkers of liver health. Both AST and ALT levelswere significantly elevated in CCl₄ administered animals for the entireduration of the study, suggesting that liver damage has occurred. Dataare mean±standard deviation (stdev); “Comp A-1”: Amino Acid CompositionA-1; p values are compared to vehicle/CCl₄ control; by one-tailed Ttest; n.s. not significant. Raw data are shown in Tables 29 and 30.

TABLE 29 ALT level results Liver ALT Vehicle/ Vehicle/ Comp A-1, CompA-1, Comp A-1, Sham CCL4 23 mg/ml 76 mg/ml 153 mg/ml mean 1608.4 4153.43694.9 3023.4 2992.7 stdev 1099.5 1427.4 2106.4 1343.8 1674.2 n.s. p <0.05 p = 0.0371

TABLE 30 AST level results Liver AST Vehicle/ Vehicle/ Comp A-1, CompA-1, Comp A-1, Sham CCL4 23 mg/ml 76 mg/ml 153 mg/ml mean 155.8 933.6879.2 554.7 680.4 stdev 69.7 237.0 527.3 336.6 431.2 n.s. p < 0.01 p =0.0394

Summary

Treatment with Amino Acid Composition A-1 resulted in reduction ofchemically-induced fibrosis as indicated by reduced levels ofhydroxyproline, a marker for collagen production, and in improvement ofclinical biomarkers of liver damage as indicated by reduction in levelsof liver enzymes ALT and AST (Tables 31-33).

TABLE 31 Hepatic Hyp content level results: raw data HydroxyprolineVehicle/ Comp A-1, Comp A-1, Comp A-1, Sham Vehicle/CCL4 23 mg/ml 76mg/ml 153 mg/ml 0.122 0.241 0.246154 0.190323 0.248649 0.277 0.3180.529578 0.174684 0.24 0.152 0.298 0.234783 0.226549 0.18 0.108 0.4930.216393 0.169128 0.174233 0.123 0.2 0.294737 0.175887 0.133333 0.1080.196 0.22439 0.107692 0.135758 0.232 0.183 0.305512 0.212389 0.2102190.177 0.393064 0.316191 0.150265 0.272897 0.612174 0.231293 0.1926830.18018 0.308824 0.164341 0.218803 0.203279 0.17971

TABLE 32 ALT level results: raw data Liver ALT Vehicle/ Comp A-1, CompA-1, Comp A-1, Sham Vehicle/CCL4 23 mg/ml 76 mg/ml 153 mg/ml 685.07374963.448 1299.647 4325.237 2611.524 2623.343 578.7053 5069.816 4325.2372150.594 1606.933 5235.278 5566.202 2304.237 1866.945 3805.214 2115.1385188.003 1051.454 696.8924 779.6234 4384.331 3828.851 1488.746 1725.121637.7988 4207.05 330.5123 4313.419 3722.483 1417.834 5471.652 649.61764112.501 5211.641 1311.466 5105.273 1441.471 2859.717 4797.986 3462.4715495.29 2564.249 1216.916 4147.957 4892.536 5318.009 1796.033 5436.1965329.828 2836.079 5069.816 3852.489 5247.097 2457.881 5046.179 5034.361346.922

TABLE 33 AST level results: raw data Liver AST Vehicle/ Comp A-1, CompA-1, Comp A-1, Sham Vehicle/CCL4 23 mg/ml 76 mg/ml 153 mg/ml 95.37346908.3081 315.7015 703.1751 508.1721 57.38585 1050.129 928.5682 720.9027335.9616 239.7263 877.918 1389.484 371.4167 379.0142 194.1412 660.12241047.596 262.5189 211.8688 123.231 599.3423 589.2123 267.5839 510.7046102.971 675.3175 181.4787 819.6704 885.5156 237.1938 1470.525 285.3115629.7324 1214.742 196.6737 1070.389 305.5715 414.4693 941.2307 733.56511690.853 505.6396 252.3889 976.6858 1100.779 1485.72 297.974 1088.1161232.469 356.2217 1437.602 918.4382 1483.187 406.8718 1189.416 1108.376267.5839

Example 5 Therapeutic Treatment with Amino Acid Composition A-1 ImprovesOral Glucose Tolerance in a Pre-Clinical Animal Model

Amino Acid Composition A-1 and metformin were tested for their abilityto affect glucose tolerance in a genetically obese B6.Cg-Lep^(ob)/J(ob/ob) mouse model (Maida A, et al., 2010, PMID: 20972533).

Model Description

B6.Cg-Lep^(ob)/J (ob/ob) mice harbor a spontaneous mutation of leptin(Lep) gene. ob/ob mice exhibit hyperphagia, obesity, and metabolicsyndrome/T2DM-like symptoms, e.g. hyperglycemia, hyperinsulinemia, andinsulin resistance. ob/ob mice have impaired intestinal barrierfunction, gut microbial translocation, and an inflammatory, fibrogenicphenotype of hepatic stellate cells (Brun P et al., 2004, PMID:17023554). ob/ob mice develop skeletal muscle hypoplasia in quadricepsfemoris, similar to the effect of aging in humans (Hamrick M W et al.,2004, PMID: 15003785). ob/ob mice exhibit intolerance to glucose andinsulin. Metformin lowers plasma glucose (Cool B, et al., Cell Metab2006, PMID: 16753576), liver triglyceride, and reverses NAFLD in ob/obmice (Lin H Z et al., 2000, PMID: 10973319; Cool B, et al., Cell Metab2006, PMID: 16753576). A single dose of metformin treatment reducesblood glucose and improves glucose tolerance (OGTT) in C57/BL6.

Experimental Design

Eight-week-old male ob/ob mice were subjected to treatment of testarticles (Amino Acid Composition A-1 and metformin) followed by oralglucose tolerance test (OGTT) on Day 3. Mice were randomized by bodyweight and unfasted blood glucose on Day −1. Body weight was recordeddaily in the morning before AM dosing on Day 1, Day 2, and Day 3. Testarticles were dosed by oral gavage at 10 ml/kg. Dosage of a test articlewas calculated based on daily body weight. Treatment schedule and doseare listed in the following section (Table 34). AM doses wereadministered at 0700, and PM doses were administered at 1800. Oralglucose tolerance test (OGTT) was performed after 6-hour fasting on Day3.

TABLE 34 Treatment schedule Group Test article # Dosing Schedule Group 1Vehicle N = 5 Vehicle dosed on Day 1 and Day 2 at 0700 and 1800, and Day3 at 0700 and 30 min before OGTT for a total of 6 doses. Group 2Metformin N = 5 Metformin hydrochloride (450 mg/kg, QD PO at thebeginning of dark cycle) dosed on Day −1, Day 1, and Day 2 at 1800, andat 30 min before OGTT on Day 3 for a total of 4 doses. Group 3 AminoAcid N = 5 Amino Acid Composition A-1 (1500 mg/kg, BID PO at Composition0700 and 1800) dosed on Day 1 and Day 2 at 0700 and 1800, A-1 and Day 3at 0700 and 30 min before OGTT for a total of 6 doses. Group 4 AminoAcid N = 5 Amino Acid Composition A-1 (3000 mg/kg, BID PO at Composition0700 and 1800) dosed on Day 1 and Day 2 at 0700 and 1800, A-1 and Day 3at 0700 and 30 min before OGTT for a total of 6 doses.

Baseline Glucose and Biochemistry (Insulin, Triglyceride andCholesterol)

Mice were fasted for 6 hours prior to OGTT test. Food was removed at0700 hours on Day 3; water was provided during fasting. Blood sampleswere collected from tail snip or facial puncture at −30 min (relative toOGTT) into K₂EDTA tubes for baseline glucose and blood biochemistry(insulin, triglyceride, and cholesterol). Blood glucose was measured bya glucometer (SDI StatStrip Xpress or equivalent). Plasma was collectedin K₂EDTA and saved at −80° C.

Oral Glucose Tolerance Test (OGTT)

Mice were bled for baseline glucose and plasma at −30 min. Test articleswere then dosed by oral gavage at −30 min. Glucose was administered peros (P.O.) at a dosage of 2.0 g/kg body weight. Blood glucose levels weremeasured at 0 min immediately prior to glucose injection and then at 15,30, 60, 120 and 240 minutes thereafter (shown as 0.25, 0.5, 1, 2, and 4hours in Table 35 below).

Results are shown in Table 35. Data are mean±standard deviation (stdev).(p values by Dunnett's multiple comparisons: **p<0.005 compared tovehicle control; ***p<0.001 compared to vehicle control; ****p<0.0005compared to vehicle control.)

Results

TABLE 35 OGTT results: Mean Blood glucose levels (mg/dl) and standarddeviations (stdev) Amino Acid Amino Acid Comp A-1, Comp A-1, Vehicle1500 mg/kg 3000 mg/kg Metformin mean blood mean blood mean blood meanblood glucose glucose glucose glucose Timepoint level level level level(hours) (mg/dl) stdev (mg/dl) stdev (mg/dl) stdev (mg/dl) stdev −1 241.8108.3 245.6 89.4 229.6  78.2 196.4   59.8 0 282.6 47.0 374.6 97.6 303.0 77.1 199.4   62.6 0.25 655.0 107.2 575.6 73.8 456.2** 36.6 353.6****73.6 0.5 640.6 92.6 555.2 84.0 513.0  47.9 390.2***  99.5 1 378.0 111.1386.6 27.5 316.4  86.1 317.6   116.9 2 236.6 54.8 243.5 18.4 230.0 101.1 158.2   44.0 4 197.8 53.3 214.8 56.8 179.8  81.3 109.4   29.0

Summary

Treatment with Amino Acid Composition A-1 resulted in improvement oforal glucose tolerance, as indicated by improved blood glucose clearanceupon oral glucose loading. In addition, 3-day treatment with Amino AcidComposition A-1 did not alter baseline blood glucose in ob/ob mice(Table 36).

TABLE 36 OGTT results: Blood glucose levels (mg/dl) raw data Timepoint(hours) Vehicle −1 211 141 211 219 427 0 239 256 273 284 361 0.25 741676 514 578 766 0.5 551 621 604 630 797 1 305 317 327 369 572 2 182 243230 203 325 4 146 203 167 188 285 Timepoint (hours) Amino Acid Comp A-1,1500 mg/kg −1 224 220 190 191 403 0 331 548 347 316 331 0.25 526 702 580531 539 0.5 532 419 621 587 617 1 431 365 367 395 375 2 246 243 220 2654 192 193 175 299 Timepoint (hours) Amino Acid Comp A-1, 3000 mg/kg −1137 294 179 214 324 0 242 359 203 329 382 0.25 412 490 438 443 498 0.5513 482 467 512 591 1 240 351 220 342 429 2 161 334 148 160 347 4 120235 110 139 295 Timepoint (hours) Metformin −1 265 248 183 120 166 0 274234 220 129 140 0.25 387 397 427 310 247 0.5 462 439 482 307 261 1 365399 431 239 154 2 154 183 219 124 111 4 118 93 155 101 80

Example 6 Therapeutic Treatment of NAFLD, NASH, and HCC with Amino AcidComposition A-1 in a Pre-Clinical Animal Model

Amino Acid Composition A-1 and Obeticholic acid(6α-ethyl-chenodeoxycholic acid; “OCA”) were tested for their ability totreat NASH in the STAM™ model (Stelic Institute & Co., Tokyo, Japan;Saito K. et al., 2015 Sci Rep 5: 12466). Two additional groups of normalC57BL/6 mice fed standard chow and vehicle treated STAM™ mice wereincluded as controls. All animals receiving treatment or vehicle weretreated starting at 6 weeks until 9 weeks of age. Compounds wereadministered via oral gavage, with a dose volume of 10 ml/kg. Amino AcidComposition A-1 was administered twice daily at a dose of 1500 mg/kg,and OCA was administered once daily at a dose of 30 mg/kg.

STAM™ Mouse Model Description

STAM™ is a model for non-alcoholic steatohepatitis (NASH) andhepatocellular carcinoma (HCC), developed by SMC Laboratories, Inc. andcreated by the combination of chemical and dietary interventions usingC57BL/6 mice (Saito K. et al., 2015 Sci Rep 5: 12466). Mice are treatedwith a low dose of streptozotocin at birth and fed a high fat dietstarting at 4 weeks. Evidence of fatty liver is present by 5 weeks,followed by NASH by 7 weeks and fibrosis by 9 weeks.

Induction of NASH

NASH was induced in 53 male mice by a single subcutaneous injection of200 μg streptozotocin (STZ, Sigma-Aldrich, USA) solution 2 days afterbirth and feeding with high fat diet (HFD, 57 kcal % fat, Cat# HFD32,CLEA Japan, Japan) after 4 weeks of age.

Route of drug administration, preparation of dosing solutions andtreatment doses

Amino Acid Composition A-1, OCA and Vehicle (described below) wereadministered by oral route in a volume of 10 mL/kg. Amino AcidComposition A-1 was solubilized in deionized water to 150 mg/ml (10×).OCA (Advanced ChemBlocks Inc.) was resuspended in 0.5% methycellulose inwater to 3 mg/ml (10×). Amino Acid Composition A-1 was administered at adose of 1500 mg/kg twice daily (9 am and 7 pm). OCA was administered ata dose of 30 mg/kg once daily (9 am).

Histological Analyses

Liver samples from mice in Group 2 (Vehicle), 3 (Amino Acid CompositionA-1) and 4 (OCA) were used for the following assays. For HE staining,sections were cut from paraffin blocks of liver tissue prefixed inBouin's solution and stained with Lillie-Mayer's Hematoxylin (Muto PureChemicals Co., Ltd., Japan) and eosin solution (Wako Pure ChemicalIndustries). NAFLD Activity score (NAS) was calculated according to thecriteria of Kleiner (Kleiner D. E. et al., Hepatology, 2005; 41:1313).

Experimental Design

Study Groups

Group 1: STZ: Ten neonatal STZ-primed mice were fed with a normal dietad libitum without any treatment until 9 weeks of age.

Group 2: Vehicle: Ten NASH mice were orally administered vehicle (10%phosphate buffered saline, pH 7.2) in a volume of 10 mL/kg twice daily(9 am and 7 pm) from 6 to 9 weeks of age.

Group 3: Amino Acid Composition A-1: Ten NASH mice were orallyadministered water for irrigation supplemented with Amino AcidComposition A-1 at a dose of 1500 mg/kg twice daily (9 am and 7 pm) from6 to 9 weeks of age.

Group 4: OCA: Ten NASH mice were orally administered 0.5%methylcellulose supplemented with OCA at a dose of 30 mg/kg once daily(9 am) from 6 to 9 weeks of age.

Group 5: Normal: Ten normal mice were fed with a normal diet ad libitumwithout any treatment until 9 weeks of age.

Group 6: HFD: Ten normal mice were fed with a high fat diet ad libitumwithout any treatment until 9 weeks of age.

Histological Analysis Results: HE Staining, NAFLD Activity Score andα-Smooth Muscle Actin Staining

Non-alcoholic fatty liver disease activity score results

The non-alcoholic fatty liver disease (NAFLD) activity score wasassessed via histological analysis and grading of H&E stained liversections from each animal. This score is the sum of three individualscores that grade the degree of steatosis (0-3), inflammation (0-2), andhepatocyte ballooning (0-2). All tissues were graded using the scoringcriteria of Kleiner et al. (Kleiner et al. Hepatology. 2005; 41(6):1313-21). Results are shown in Table 37. Data are mean±standarddeviation (stdev). Normal C57BL/6 mice fed standard chow had a meanscore of 0+/−0. Vehicle treated STAM™ mice had a mean score of4.7+/−0.67. Amino Acid Composition A-1 treated mice had a mean score of3.1+/−0.74. OCA treated mice had a mean score of 2.9+/−0.74. Both AminoAcid Composition A-1 and OCA were statistically different from vehiclefor NAFLD Activity Score when compared using Dunnett's multiplecomparisons test (Amino Acid Composition A-1 p=0.0001, OCA p=0.0001).

Similarly, Amino Acid Composition A-1 treated mice showed a meanballooning score of 0.4+/−0.52, compared to a mean ballooning score forvehicle treated STAM™ mice of 1.6+/−0.52, and a mean ballooning scorefor OCA treated mice of 0.3+/−0.48. Both Amino Acid Composition A-1 andOCA were statistically different from vehicle for ballooning score whencompared using Dunnett's multiple comparisons test (Amino AcidComposition A-1 p=0.0001, OCA p=0.0001). Raw data are shown in Tables37-40.

TABLE 37 NAFLD Activity Score NAFLD Activity Score (NAS) Normal Vehicle-Amino Acid C57BL/6 treated Composition A-1 OCA treated Condition miceSTAM mice treated STAM mice STAM mice Mean 0 4.7 3.1 2.9 stdev 0 0.670.74 0.74

TABLE 38 NAFLD Activity: Steatosis Score Steatosis Normal Vehicle- AminoAcid C57BL/6 treated Composition A-1 OCA treated mice STAM mice treatedSTAM mice STAM mice Mean 0 1 0.9 0.8 stdev 0 0.00 0.32 0.42

TABLE 39 NAFLD Activity: Inflammation Score Inflammation Normal Vehicle-Amino Acid C57BL/6 treated STAM Composition A-1 OCA treated mice micetreated STAM mice STAM mice Mean 0 2.1 1.8 1.8 stdev 0 0.32 0.63 0.79

TABLE 40 NAFLD Activity: Ballooning Score Ballooning Normal Vehicle-Amino Acid C57BL/6 treated Composition A-1 OCA treated mice STAM micetreated STAM mice STAM mice Mean 0 1.6 0.4 0.3 stdev 0 0.52 0.52 0.48

Fibrosis: Sirius Red Staining Results

Fibrosis was assessed by analysis of Sirius red positively stained cellarea from stained liver sections from each animal. Images werequantified using the percent of positively stained area was used as ameasure of fibrosis. Results of this analysis are shown in Table 39.Data are mean±standard deviation (stdev). Normal C57BL/6 mice fedstandard chow had a mean positive area of 0.286+/−0.09. Vehicle treatedSTAM™ mice had a mean positive area of 1.1+/−0.26. Amino AcidComposition A-1 treated mice had a mean positive area of 0.828+/−0.33.OCA treated mice had a mean score of 0.776+/−0.25. Amino AcidComposition A-1 and OCA were statistically different from vehicle whencompared using Dunnett's multiple comparisons test (Amino AcidComposition A-1 p=0.00494, OCA p<0.016). Raw data are shown in Table 41.

TABLE 41 Fibrosis (mean positively stained area, Sirius red) NormalVehicle- Amino Acid C57BL/6 treated Composition A-1 OCA treatedCondition mice STAM mice treated STAM mice STAM mice Mean 0.286 1.10.828 0.776 stdev 0.09 0.26 0.33 0.25

Similarly to the statistically significant improvement in the NAFLDactivity score, ballooning, and fibrosis in the STAM mouse model aftertreatment with Amino Acid Composition A-1 (FIG. 1A), a statisticallysignificant improvement in the NAFLD activity score, ballooning, andfibrosis was determined in the high-fat, high fructose and cholesteroldiet (HFFC) mouse model after treatment with Amino Acid Composition A-1(FIG. 1B).

α-Smooth Muscle Actin (α-SMA) Staining Results

Liver sections of all mice were stained for the marker α-smooth muscleactin (αSMA) to identify activated hepatic stellate cells. Images werequantified using the percent of positively stained area was used as ameasure of stellate cell activation. Results are shown in Table 42. Dataare mean±standard deviation (stdev); p values are compared tovehicle-treated STAM mice control; by one-tailed T test.

Normal C57BL/6 mice fed standard chow had a mean positive area of0.682+/−0.26. Vehicle treated STAM™ mice had a mean positive area of2.128+/−0.50. Amino Acid Composition A-1 treated mice had a meanpositive area of 1.657+/−0.84. OCA treated mice had a mean score of1.562+/−0.31.

TABLE 42 Activated hepatic stellate cells (mean positively stained area,α-smooth muscle actin) Normal Vehicle- Amino Acid C57BL/6 treatedComposition A-1 OCA treated Condition mice STAM mice treated STAM miceSTAM mice Mean 0.682 2.128 1.657 1.562 stdev 0.26 0.50 0.84 0.31 p =0.073 p < 0.05

Summary

Treatment with Amino Acid Composition A-1 significantly reduced NASHseverity to levels equivalent to Farnesoid X Receptor (FXR) inhibitionby OCA (which is currently under clinical investigation by InterceptPharmaceuticals, Inc. for treatment of NASH), as indicated bysignificant reduction in NAFLD Activity Score (NAS) (mean NAS:3.1+/−0.74 for Amino Acid Composition A-1 vs. vehicle treated STAM™ micemean score of 4.7+/−0.67, compared to OCA treated mice mean score of2.9+/−0.74), and development of fibrosis as indicated by thedownregulation of hepatic stellate cell activation (mean aSMA positivelystained area: 1.657+/−0.84 for Amino Acid Composition A-1 vs. vehicletreated STAM™ mice mean area of 2.128+/−0.50, compared to OCA treatedmice mean area of 1.562+/−0.31).

TABLE 43 NAFLD Activity Score: raw data Amino Acid NormalVehicle-treated Composition A-1 OCA treated C57BL/6 mice STAM micetreated STAM mice STAM mice 0 6 3 4 0 5 4 2 0 5 4 2 0 4 3 4 0 5 2 3 0 52 3 0 4 3 2 0 4 3 3 0 4 3 3 0 5 4 3

TABLE 44 NAFLD Activity: Steatosis Score: raw data Steatosis NormalVehicle- Amino Acid C57BL/6 treated Composition A-1 OCA treated miceSTAM mice treated STAM mice STAM mice 0 1 1 1 0 1 1 1 0 1 1 1 0 1 1 1 01 0 1 0 1 1 0 0 1 1 1 0 1 1 0 0 1 1 1 0 1 1 1

TABLE 45 NAFLD Activity: Inflammation Score: raw data InflammationNormal Vehicle- Amino Acid C57BL/6 treated STAM Composition A-1 OCAtreated mice mice treated STAM mice STAM mice 0 3 1 2 0 2 2 1 0 2 2 1 02 2 2 0 2 1 2 0 2 1 3 0 2 2 1 0 2 2 3 0 2 2 2 0 2 3 1

TABLE 46 NAFLD Activity: Ballooning Score: raw data Ballooning NormalVehicle- Amino Acid C57BL/6 treated Composition A-1 OCA treated miceSTAM mice treated STAM mice STAM mice 0 2 1 1 0 2 1 0 0 2 1 0 0 1 0 1 02 1 0 0 2 0 0 0 1 0 0 0 1 0 0 0 1 0 0 0 2 0 1

TABLE 47 Fibrosis (mean positively stained area, Sirius red): raw dataNormal Amino Acid C57BL/6 Vehicle-treated Composition A-1 OCA treatedmice STAM mice treated STAM mice STAM mice 0.26 0.79 1.07 0.36 0.35 1.430.58 0.56 0.19 1.44 0.48 1.1 0.31 1.36 0.58 1.19 0.19 1.04 1.07 0.890.36 0.75 0.34 0.91 0.24 1.07 0.86 0.66 0.37 1.13 1.43 0.72 0.18 0.830.96 0.68 0.41 1.16 0.91 0.69

TABLE 48 Activated hepatic stellate cells (mean positively stained area,α-smooth muscle actin): raw data Normal Amino Acid C57BL/6Vehicle-treated Composition A-1 OCA treated mice STAM mice treated STAMmice STAM mice 0.47 2.16 0.81 1.46 0.59 2.77 1.35 1.51 1.13 2.21 1.31.49 0.52 1.5 3.03 1.17 0.75 2.87 2.04 1.49 0.46 1.93 0.97 1.5 0.37 1.63.08 1.13 0.85 1.46 1.91 2.03 0.62 2.36 1.15 1.87 1.06 2.42 0.93 1.97

Example 7 Reduction of Fibrogenic Gene Expression in Hepatic StellateCells Treated with an Amino Acid Composition

Hepatic stellate cells in a healthy liver are in the space of Disse,between the hepatocytes and liver sinusoidal endothelial cells. Inresponse to liver injury hepatic stellate cells become activated,proliferative and contractile, increase production of αSMA, secretion oftype I and III collagens and specific MMP and TIMP proteins. LX-2 cellswere selected as a model of activated hepatic stellate cells and used totest whether specific amino acid compositions would reduce fibrogenicgene expression induced with TGFβ1.

LX-2 hepatic stellate cells (Millipore) were seeded on day 0 at 1.67E4cells per well in collagen I coated 96-well microplates (ThermoFisher)in Dulbecco's Modified Eagle Medium (DMEM, Corning) supplemented with 2%heat inactivated fetal bovine serum (HI-FBS, HyClone) and 0.2% Primocin(InVivoGen) and incubated overnight at 37° C., 5% CO2. On day 1, cellswere washed twice with 150 μL per well DPBS (Gibco) and replaced withamino acid free DMEM (US Biologicals) containing a defined custom aminoacid concentration based on the mean physiological concentrations inblood based on values published in the Human Metabolome Database (1, 2,3), with 25 mM Glucose, 1 mM Sodium Pyruvate and a dose curve of definedamino acid compositions LIVRQ+N-Acetylcysteine, LIVRQ,RQ+N-Acetylcysteine, N-acetylcysteine, LIV at 40× the concentrationpresent in the basal HMDB (Human Metabolome Database (Wishart D S, TzurD, Knox C, et al., HMDB: the Human Metabolome Database. Nucleic AcidsRes. 2007 January; 35 (Database issue):D521-6. 17202168)) derived aminoacid concentrations or individually with leucine, isoleucine, valine,arginine, glutamine or cysteine at 50× the HMDB derived concentrations.Combinations containing N-acetylcysteine were dosed with 10 mM. Cellswere pretreated for 6 hours at 37° C., 5% CO2. After pretreatment, TGβ1(R&D Systems) or vehicle was spiked into each well for a finalconcentration of 5 ng/mL and cells were incubated under this stimulusfor a further 12 hours at 37° C., 5% CO2.

After 12 hour incubation, RNA was prepared as described using theFastLane Cell Multiplex Kit (Qiagen) as described in the manufacturersprotocol. Two microliters of cell lysate was utilized in subsequentqRT-PCR experiments using the FastLane Cell Multiplex Kit at a reducedfinal qPCR reaction volume of 20 μL. Quantitative PCR was conducted onlysates to determine collagen-1a1 expression normalized to β-actinhousekeeping expression using the ΔΔCt method using TaqMan primer probes(Integrated DNA Technologies: Col1A1, Hs.PT.58.15517795; Actb,Hs.PT.39a.22214847; Acta2, Hs.PT.56a.24853961; Timp2,Hs.PT.58.14780594).

Results:

Table 49 shows the Col1a1, Acta2, and Timp2 gene expression in LX-2cells treated with amino acid combinations compared to vehicle with orwithout TGFβ1 stimulus. LIVRQ+N-Acetylcysteine, LIVRQ,RQ+N-Acetylcysteine, and N-acetylcysteine reduced Col1a1 expression andTimp2 expression. LIVRQ+N-acetylcysteine shows the largest reduction ofCol1a1, Acta2, and Timp2 gene expression. LIVRQ-N-acetylcysteine reducesActa2 expression significantly greater than N-Acetylcysteine alone,RQ+N-acetylcysteine, and LIV. LIVRQ+N-acetylcysteine reduces Timp2expression significantly greater than any of the other combinations(Table 49).

TABLE 49 Col1a1 Acta2 Timp2 Amino Number Number Number Acid Std. of Std.of Std. of TGFβ1 Supplement Mean Deviation values Mean Deviation valuesMean Deviation values Yes Vehicle 2.861 0.3151 4 0.801 0.1149 4 1.6580.2791 4 No Vehicle 1.042 0.3102 4 1.006 0.1190 4 1.022 0.2400 4 YesLIVRQNAC 1.267 0.4106 4 0.292 0.0969 4 0.535 0.0306 4 Yes LIVRQ 1.7870.2926 4 0.267 0.0637 4 0.975 0.2006 4 Yes RQNAC 1.664 0.3320 4 0.4870.1042 4 0.897 0.1932 4 Yes NAC 1.659 0.4695 4 0.647 0.1097 4 1.0760.0681 4 Yes LIV 2.831 0.3404 3 0.793 0.0812 4 1.927 0.0944 4

Table 50 shows the Col1a1 expression of individual amino acids with orwithout TGFβ1 stimulus at 1× or 50× the HMDB derived amino acidconcentration. Individually, only cysteine showed a significant decreasein Col1a1 expression at 50×.

TABLE 50 Col1a1 Amino Number Acid Std. of TGFβ1 Supplement MeanDeviation values No Vehicle 1.015 0.1832 8 Yes 1X CYS 2.491 0.1588 4 Yes50X CYS 1.695 0.3310 4 Yes 1X ILE 2.020 0.1451 4 Yes 50X ILE 2.0280.3667 4 Yes 1X LEU 1.901 0.3360 4 Yes 50X LEU 2.372 0.4153 4 Yes 1X VAL2.093 0.2157 4 Yes 50X VAL 2.203 0.5762 4 No Vehicle 1.010 0.1510 8 Yes1X ARG 1.620 0.6691 4 Yes 50X ARG 1.970 0.7740 4 No Vehicle 1.012 0.16818 Yes 1X GLN 2.340 0.7069 4 Yes 50X GLN 2.194 0.3359 4

Example 8 Reduction in Hepatocyte Inflammation after Treatment with anAmino Acid Composition

The ability of amino acids to influence hepatocyte inflammation wasassessed using HepG2 Hepatocellular Carcinoma cells stably expressingNF-kB luciferase reporter system (Signosis, Inc.). HepG2 cells wereseeded on day 0 in 4.5e4 in a 96-well microplates (ThermoFisher) inDulbecco's Modified Eagle Medium (DMEM, Corning) supplemented with 0.1%heat inactivated fetal bovine serum (HI-FBS, HyClone) and 0.2% Primocin(InVivoGen) and incubated overnight at 37° C., 5% CO2. On day 1, cellswere washed once with 150 μL per well DPBS (Gibco) and replaced withamino acid free DMEM (US Biologicals) containing a defined custom aminoacid concentration based on the mean physiological concentrations inblood based on values published in the Human Metabolome Database(Wishart D S, Tzur D, Knox C, et al., HMDB: the Human MetabolomeDatabase. Nucleic Acids Res. 2007 January; 35 (Database issue):D521-6.17202168), with 25 mM Glucose, 1 mM Sodium Pyruvate and a dose curve ofdefined amino acid compositions (i.e. vehicle, LIVRQ+N-acetylcysteine,LIVRQ, RQ+N-acetylcysteine, N-acetylcysteine alone, LIV or individuallywith Leucine, Isoleucine, Valine, Arginine, Glutamine, and Cysteine) at50× (Table 51). Cells were pretreated in the defined media for 12 hoursat 37° C., 5% CO₂. After pretreatment, TNFα (R&DSystems) or vehicle wasspiked into each well for a final concentration of 100 pM and cells wereincubated under this stimulus for an additional 6 hours at 37° C., 5%CO₂. After 12-hour incubation, cells were washed 1× in 150 ul cold PBSand lysed using Passive Lysis Buffer and luciferase assay was performedaccording to manufacturer's protocol (Signosis). Firefly luciferaseactivity was assessed using a Bio-Tek SynergyH4 plater reader andluminometer (Sitcheran R*, Comb W C, Cogswell P C, Baldwin A S*.Essential role for epidermal growth factor receptor in glutamatereceptor signaling to NF-kappaB. Mol Cell Biol. (2008) August;28(16):5061-70. Epub 2008 Jun. 9).

TNFα-stimulated NF-kB activity was unaffected by treating cells in 50×Leucine, Isoleucine, Valine, Arginine, and Glutamine, relative to the 1×Plasma amino acid baseline media. Pretreating cells in 50× Cysteine didresult in a significant blunting of TNFα-induced NF-kB activity.Combinatorial treatments with the single amino acids did have varyingeffects on the NF-kB reporter activity, but importantly, the combinationof all 6 amino acids together (LIVRQNAC) resulted in the mostsignificant inhibition of TNFα induced NF-kB activity in liver cells(Table 51).

TABLE 51 NF-kB Reporter Activity Number Amino Acid Std. of TNFαSupplement Mean Deviation values 100 pM Vehicle (1x 8865.50 333.05 2 AA)100 pM LIVRQNAC 3960.50 678.12 2 100 pM LIVRQ 5685.00 1453.81 2 100 pMRQNAC 5618.00 926.31 2 100 pM NAC 6852.00 1023.89 2 100 pM LIV 5911.00422.85 2 100 pM 1x L 5811.00 134.35 2 100 pM 50x L 6070.50 58.69 2 100pM 1x I 8129.50 713.47 2 100 pM 50x I 8937.50 17.68 2 100 pM 1x V7255.50 557.91 2 100 pM 50x V 5992.00 644.88 2 100 pM 1x R 10170.50140.71 2 100 pM 50xR 9760.00 1083.29 2 100 pM 1x Glu 8201.00 2091.62 2100 pM 50x Glu 7313.50 1054.30 2 100 pM 1x Cys 9968.50 1614.33 2 100 pM50x Cys 6820.50 23.34 2

Example 9 Treatment with an Amino Acid Composition Ameliorates NASHProgression in Two Rodent Models by Impacting Lipid Metabolism,Inflammation, and Fibrosis

The amino acid composition is formulated to simultaneously targetmultiple mechanisms of disease pathology to safely and effectively treatNASH (Table 52). As described herein, the efficacy of the amino acidcomposition was studied in two established mouse models of NASH todetermine the effect of the amino acid composition on signs and symptomsassociated with NASH and related disorders (FIG. 2).

TABLE 52 Exemplary amino acid components of the amino acid composition.Amino acid wt. ratio wt. % g/packet g dose #1 g dose #2 Leucine 1 16.781.00 g 2 g 4 g Isoleucine 0.5 8.39 0.50 g 1 g 2 g Valine 0.5 8.39 0.50 g1 g 2 g Arginine HCl 1.81 30.37 1.81 g 3.62 g   7.24 g   Glutamine 233.56 2.00 g 4 g 8 g N-acetylcysteine 0.15 2.52 0.15 g 0.3 g   0.6 g  Total amino acids 5.96 g ~12 g    ~24 g   

Animal Studies

The STAM™ mouse is a model for non-alcoholic steatohepatitis (NASH) andhepatocellular carcinoma (HCC), developed by SMC Laboratories, Inc.Evidence of fatty liver is present by 5 weeks of age, followed by NASHby 7 weeks of age, and fibrosis by 9 weeks of age. Male STAM mice weregenerated in C57BL/6 mice, which received a low dose streptozotocin 2days after birth and were fed a high fat diet (57% kcal fat, HFD32, CLEAJapan, Inc.) starting at 4 weeks old (Saito K. et al., 2015 Sci Rep 5:12466; hereby incorporated by reference in its entirety). The amino acidcomposition was administered to STAM mice at a dose of 1.6 m/kg twicedaily for 3 weeks starting at 6 weeks of age. One group of vehicletreated STAM mice was included as a control. Unfasted mice wereeuthanized at 9 weeks old. Plasma and liver samples were harvested forfurther analysis (FIG. 3).

The FATZO™ mouse is an inbred, polygenic model of obesity, metabolicsyndrome, and NASH, developed by Crown Bioscience, Inc (Peterson R G. Etal., 2017 PLoS One; hereby incorporated by reference in its entirety).Male FATZO mice were fed a high fat, fructose, and cholesterol (HFFC)diet (40% kcal fat, D12079B, Research Diets, Inc. and 5% fructose indrinking water) starting at 6 weeks old to induce NAFLD and NASH.Evidence of fatty liver is present by 4 weeks post induction, followedby NASH by 16 weeks post induction and fibrosis by 20 weeks ofinduction. The designed amino acid composition was administered at adose of 3.0 g/kg twice daily for 4 weeks starting at 16 weeks postinduction (FIG. 3). One group of vehicle treated FATZO mice was includedas control. Unfasted mice were euthanized at 20 weeks post-induction.Plasma and liver samples were harvested for further analysis.

Histological Analysis

The Aperio ScanScope CS whole slide digital imaging system (Vista,Calif.) was used for imaging in H&E, Picric Sirius Red, SMA, F4/80.Images were captured from whole slides.

The livers were evaluated by veterinary pathologists blind to sample IDusing the NASH Clinical Research Network (CRN) liver histologicalscoring system (Kleiner D E, et al., 2015, hereby incorporated byreference in its entirety). The NASH CRN Scoring System assessesprogression of steatosis, lobular inflammation, hepatocyte ballooning,degeneration, and fibrosis. One cross section of liver for each case wasanalyzed with the NASH score system. Steatosis, lobular inflammation,and fibrosis progression was assessed on a 0-3 scale. Ballooningdegeneration was assessed on a 0-2 scale.

The Positive Pixel Count algorithm of the Aperio Automatic ImageQuantitation was used to quantify the percentage of a specific stainpresent in a scanned slide image. A range of color (range of hues andsaturation) and three intensity ranges (weak, positive, and strong) weremasked and evaluated. The algorithm counted the number and intensity-sumin each intensity range, along with three additional quantities: averageintensity, ratio of strong/total number, and average intensity of weakpositive pixels.

A specific positive pixel algorithm was used for imaging the Sirius Redand Oil Red 0 liver sections. The positive pixel algorithm was modifiedto distinguish between the orange and blue colors. Alterations from thenormal “hue value” (0.1 to 0.96) and “color saturation” (0.04 to 0.29),were made for the Sirius Red evaluation. Vasculature and artifacts wereexcluded from analysis.

Liver Triglyceride and Cholesterol Measurement

Liver total lipid-extracts were obtained by Folch's method (Folch J. etal., J. Biol. Chem. 1957; 226: 497; hereby incorporated by reference inits entirety). Liver samples were homogenized in chloroform-methanol(2:1, v/v) and incubated overnight at room temperature. After washingwith chloroform-methanol-water (8:4:3, v/v/v), the extracts wereevaporated to dryness, and dissolved in isopropanol. Liver triglycerideand cholesterol contents were measured by the Triglyceride E-test andCholesterol E-test, respectively.

Gene Expression Analysis

Liver RNA samples were converted into cDNA libraries using the IlluminaTruSeq Stranded mRNA sample preparation kit (Illumina # RS-122-2103).Transcriptome were analyzed at Q2 Solutions (Morrisville, N.C.). RNA Seqdata were normalized and analyzed using Ingenuity Pathway Analysis(QIAGEN Bioinformatics). Mouse liver gene expression at the pathwaylevel was focused on because it is translatable to human NAFLD (TeufelA, et al., Gastroenterology, 2016, hereby incorporated by reference inits entirety).

Metabolite Analysis

Metabolic profiling based on both capillary electrophoresistime-of-flight mass spectrometry (CE-TOFMS) and LC-TOFMS platforms wasperformed at Human Metabolome Technologies (Yamagata, Japan).Metabolites in the samples were identified by comparing the migrationtime and m/z ratio with authentic standards and quantified by comparingtheir peak areas with those of authentic standards.

Liver Cytokine/Chemokine Measurement

The levels of IL-1b, MCP-1, and MIP-1 protein in liver were quantifiedusing the multiplex ELISA Assay (Meso Scale Discovery, Rockville, Md.).

The Amino Acid Composition Improves Ballooning and Fibrosis in Both STAMand FATZO Mice

Treatment with the amino acid composition significantly reduced NAFLDactivity scores (NAS) in both STAM and FATZO mice (FIG. 4A). Treatmentwith the amino acid composition also significantly decreased hepatocyteballooning in STAM mice (FIG. 4B). Scores of steatosis and inflammationwere not changed according to histological measures by treatment of STAMmice with the amino acid composition. The Sirius Red-positive, fibrosisarea was significantly lowered by treating the STAM mice with the aminoacid composition, while the Oil Red O area was not changed by treatingthe STAM mice with the amino acid composition (FIG. 4C). Livertriglyceride and cholesterol levels were not changed.

Treatment with the amino acid composition also significantly decreasedhepatocyte ballooning in FATZO mice (FIG. 4D). Scores of steatosis andinflammation as well as liver triglyceride and cholesterol levels werenot changed in the FATZO mice treated with the amino acid compositiontreatment. The Sirius Red-positive, fibrosis area was significantlylowered by treatment of the FATZO mice with the amino acid composition,while the Oil Red O area was not changed by treatment of the FATZO micewith the amino acid composition treatment (FIG. 4E).

The Amino Acid Composition Enhances Fatty Acid Oxidation

NAFLD is characterized by hepatic lipid accumulation. Liver triglycerideis attributable to a precise balance between acquisition by de novolipogenesis and uptake of non-esterified fatty acids from the plasma,versus disposal by fatty acid oxidation and by the secretion oftriglyceride-rich lipoproteins (Kawano Y, Cohen D E, J Gastroenterol.2013, hereby incorporated by reference in its entirety). Compared tocontrol mice, STAM mice had higher liver unsaturated fatty acids, whichwere reduced by treatment with the amino acid composition (FIG. 5A andTable 53). Liver acylcarnitines in STAM mice were increased by treatmentwith the amino acid composition, suggesting enhanced fatty acidbeta-oxidation (FIG. 5B and Table 53).

TABLE 53 P-values and fold changes for liver acylcarnitine andunsaturated fatty acids following treatment of STAM mice with the aminoacid composition (treated) compared to control. Control Treated Controlfold Treated fold Lipid KEGG ID HMDB ID p-val change p-val changeAC(13:1) No ID No ID −1.61 7.94E−02 1.32 FA(14:3) No ID No ID 5.17E−031.69 4.83E−01 −1.24 FA(20:3) No ID No ID 6.97E−06 18.29 1.35E−01 −2.17FA(22:4) No ID No ID 7.12E−07 34.79 3.15E−01 −1.79 FA(22:5)-1 No ID NoID 2.39E−02 3.31 1.19E−01 −1.80 FA(22:5)-2 No ID No ID 2.11E−04 3.031.69E−02 −1.92 Linoleic acid C01595 HMDB00673 9.90E−04 3.18 1.68E−02−1.77 Linolenic acid C06427 HMDB01388 3.57E−05 35.88 4.08E−02 −1.99Oleic acid C00712 HMDB00207 1.95E−04 18.05 3.67E−02 −1.88 Palmitoleicacid C08362 HMDB03229 9.84E−05 2.69 2.70E−02 −1.48

Differential gene expression patterns in the liver impacted by treatmentwith the amino acid composition were interpreted in the context of theupstream regulator systems biology knowledgebase framework developed byIngenuity Pathway Analysis. Computed z-scores indicated that the geneexpression patterns are consistent with activation of ACOX1, whichencodes peroxisomal fatty acid oxidation, as an upstream regulator (FIG.6 and Table 54).

TABLE 54 P-values and fold changes for gene expression associated withthe ACOX1 pathway following treatment of STAM mice with the amino acidcomposition (treated) compared to control. IPA_ IPA_ Control Treatedupstream_ ACOX1_ gene_ fold Control fold Treated gene regulator pathname change p-val change p-val Akr1c6 ACOX1 ACOX1 AKR1C4 −1.68 1.88E−061.207 8.606E−02 C9 ACOX1 ACOX1 −3.10 7.81E−07 1.370 1.678E−02 Ces3aACOX1 ACOX1 −2.10 2.69E−06 1.379 3.900E−02 Ces3b ACOX1 ACOX1 −3.161.05E−07 1.476 8.274E−02 Cyp2c50 ACOX1 ACOX1 Cyp2c54 −1.72 1.24E−041.243 6.999E−02 Cyp4a12a ACOX1 ACOX1 −1.59 4.60E−03 1.293 8.589E−02Cyp7b1 ACOX1 ACOX1 −4.45 4.29E−04 1.408 7.877E−02 Egfr NFKB; ACOX1 −1.981.31E−04 1.348 1.187E−02 ACOX1 Gstp1 ACOX1 ACOX1 −2.31 2.56E−06 1.2812.924E−02 Mup1 ACOX1 ACOX1 −7.69 1.47E−03 1.781 7.683E−02 Mup11 ACOX1ACOX1 −2.47 9.01E−03 1.703 5.779E−02 Mup14 ACOX1 ACOX1 −2.05 1.27E−021.395 4.890E−02 Mup16 ACOX1 ACOX1 −6.27 4.38E−03 1.465 7.558E−02 Mup6ACOX1 ACOX1 −1.73 2.27E−02 1.330 5.784E−02 Selenbp2 TGFB; IL10; ACOX1Selenbp1 −15.77 3.73E−05 3.015 2.916E−02 ACOX1 Serpina1c TGFB; ACOX1−2.25 7.22E−09 1.290 5.612E−02 ACOX1 Serpina1e TGFB; ACOX1 −43.203.93E−08 2.361 1.852E−02 ACOX1 Slc4a4 ACOX1 ACOX1 1.55 7.00E−06 −1.2094.682E−03 Trib3 IL2; NFKB; ACOX1 2.40 3.14E−04 −1.472 1.987E−02 ACOX1

The Amino Acid Composition Tempers Inflammation Pathways

Inflammation is a “second-hit” of NASH. The differential gene expressionpatterns in the liver as a result of treatment with the amino acidcomposition yielded z-scores within IPA analysis associated withupstream regulator activation of anti-inflammatory IL-10 (FIG. 7A) andinhibition of pro-inflammatory NF-kB (FIG. 7B and Table 55),interferons, IL-1b, and IL-2 (FIG. 7C and Table 55). At the proteinlevel, treatment with the amino acid composition significantlydown-regulated hepatic MCP-1 and MIP-1, which are the ligands of C—Cchemokine receptor types 2 (CCR2) and 5 (CCR5), respectively (FIG. 8).Thus, treatment with the amino acid composition tempered the immunesystem toward an anti-inflammatory state, which may dampen NASHprogression.

TABLE 55 P-values and fold changes for gene expression associated withthe ACOX1 pathway following treatment of STAM mice with the amino acidcomposition (treated) compared to control. Control Treated IPA upstreamIPA gene IL10_ IL1b_ IL2 NFKB_ TGFB_ fold Control fold Treated generegulator name path path path path path change p-val change p-val Abcb1aNFKB; IL10 IL10 NFKB 3.02 1.55E−06 −1.239 1.047E−01 Abcb1b NFKB; IL10IL10 NFKB −2.24 4.90E−03 1.272 9.745E−02 Acta1 TGFB TGFB 7.96 5.87E−03−1.849 1.044E−01 Adora1 TGFB TGFB 1.94 1.99E−05 −1.222 3.262E−02AK007436 NFKB ADAMTS9 NFKB 2.32 6.38E−03 −1.788 6.451E−02 AK043676 IL1bPFKP IL1b 1.68 3.12E−03 −1.462 1.586E−02 AK154184 IL1b; TGFB CYBA IL1bTGFB 1.99 1.02E−03 −1.222 9.247E−02 AK158038 IL2 NAV1 IL2 1.62 2.63E−02−1.659 1.587E−02 Atf5 IL1b; IL2 IL1b IL2 1.71 3.00E−04 −1.223 9.622E−02Bcl2a1d IL1b; IL2; NFKB IL1b IL2 NFKB 4.00 1.74E−04 −1.733 3.064E−02Capn5 IL2 IL2 1.51 2.05E−05 −1.235 5.908E−03 Ccrl IL1b; IL2; TGFB IL1bIL2 TGFB 2.82 1.28E−03 −1.352 7.496E−02 Cd274 IL1b; IL2; NFKB IL1b IL2NFKB 2.37 1.71E−07 −1.282 1.506E−02 Cd83 IL1b; IL2; NFKB; IL1b IL2 NFKBTGFB 2.41 5.36E−07 −1.434 6.661E−03 TGFB Chst11 TGFB TGFB 2.91 7.87E−06−1.308 1.881E−02 Clec2i TGFB TGFB 1.80 1.01E−03 −1.226 5.963E−02 EgfrNFKB NFKB −1.98 1.31E−04 1.348 1.187E−02 Entpd1 IL2 IL2 1.87 1.84E−04−1.218 8.413E−02 Fgf21 TGFB TGFB 49.56 1.03E−03 −1.478 4.472E−02 GabrdTGFB TGFB 5.82 1.48E−04 −1.511 5.978E−02 Gbp4 IL1b; IL10 Gbp6 IL10 IL1b1.60 1.01E−03 −1.234 5.234E−02 Gbp5 IL10 IL10 1.81 7.82E−05 −1.2176.120E−02 Gm8909 IL1b; NFKB; IL10 HLA-A IL10 IL1b NFKB 4.03 8.19E−04−1.943 2.569E−02 Gpr85 TGFB TGFB 2.46 1.77E−02 −1.685 6.628E−02 Gucy2cTGFB TGFB 2.35 8.97E−03 −1.395 1.080E−01 Hk2 IL1b; IL2 IL1b IL2 2.002.01E−04 −1.301 9.834E−02 Hsd17b6 TGFB TGFB 2.89 4.20E−05 −1.2441.036E−02 Il1rn IL1b; NFKB; IL10 IL1b NFKB TGFB 4.43 5.94E−09 −1.2731.951E−02 TGFB; IL10 Lama3 IL1b IL1b −3.09 1.90E−05 1.650 5.542E−02 LckIL2 IL2 1.92 8.87E−04 −1.246 3.027E−02 Lifr IL1b; IL2; TGFB IL1b IL2TGFB −4.43 2.43E−05 1.406 2.099E−02 Msr1 TGFB TGFB 1.58 1.13E−03 −1.2265.836E−02 Mst1r TGFB TGFB 2.08 4.17E−03 −1.523 1.147E−02 Nlrp3 TGFB TGFB1.93 1.30E−03 −1.506 2.456E−02 P2ry14 TGFB TGFB 3.29 4.67E−05 −1.2684.039E−02 Pcsk1 IL1b IL1b 2.07 9.00E−03 −2.070 8.150E−02 Pla2g4a IL1b;TGFB IL1b TGFB 2.10 7.94E−05 −1.265 1.064E−01 Plb1 IL1b IL1b 2.012.09E−02 −1.696 2.324E−02 Rgs16 IL1b; IL2; NFKB IL1b IL2 NFKB 7.682.47E−05 −1.604 4.319E−02 Saa4 IL1b IL1b −1.72 4.76E−02 1.283 9.771E−02Selenbp2 TGFB; IL10 Selenbp1 IL10 TGFB −15.77 3.73E−05 3.015 2.916E−02Sema3b TGFB TGFB 4.12 4.78E−05 −1.285 9.461E−02 Serpina1c TGFB TGFB−2.25 7.22E−09 1.290 5.612E−02 Serpina1e TGFB TGFB −43.20 3.93E−08 2.3611.852E−02 Serpina3k IL1b; NFKB; TGFB IL1b NFKB TGFB −2.95 1.85E−08 1.5001.125E−02 Serpinb2 IL1b; NFKB IL1b NFKB 1.94 4.98E−02 −1.935 1.865E−02Slc23a2 TGFB TGFB 2.00 1.02E−05 −1.258 2.229E−02 Slc2a6 NFKB; IL10 IL10NFKB 1.79 2.68E−02 −1.281 9.775E−02 Slc7a1 NFKB; TGFB NFKB TGFB 1.643.96E−03 −1.324 8.657E−02 Slc7a11 IL1b; IL10 IL10 IL1b 65.45 1.35E−03−1.869 1.068E−01 Tk1 IL1b IL1b −2.40 4.99E−06 1.295 4.036E−02 Tlr11 IL10IL10 1.66 6.11E−03 −1.368 6.005E−02 Tlr2 IL1b; IL2; NFKB; IL10 IL1b IL2NFKB TGFB 2.12 5.32E−05 −1.300 5.430E−02 TGFB; IL10 Trib3 IL2; NFKB IL2NFKB 2.40 3.14E−04 −1.472 1.987E−02 Xcl1 IL2 IL2 2.52 1.22E−03 −1.7966.279E−02

The Amino Acid Composition Prevents Fibrogenesis Pathways

Fibrosis is at the nexus of several biologic processes, such asmetabolic dysregulation, inflammation, and cell death. Lipidaccumulation in hepatocytes and chronic inflammation induce fibrogenicactivation of hepatic stellate cells (Wobser H, et al., Cell Res. 2009,which is hereby incorporated by reference in its entirety). The livergene expression pattern resulting from treatment with the amino acidcomposition was consistent with the suppression of the fibrogenic TGF-bsignaling pathway (FIG. 7D).

Increasing evidence implicates that CCR2/CCR5 and their ligands,including MCP-1/MIP-1, promote macrophage recruitment and hepaticstellate cell activation which contribute to fibrosis following livertissue damage (Lefebvre E, et al., PLoS One 2016, which is herebyincorporated by reference in its entirety). The amino acid compositiondisplayed a potent antifibrotic activity in the STAM model of NASH viareducing hepatic TGF-b signaling and MCP-1 and MIP-1 proteins (FIG. 8).

Conclusion

The amino acid composition demonstrated consistent disease modifyingactivity in both STAM and FATZO mouse models of NASH includingimprovement in NAS and amelioration of ballooning and fibrosis. Theactivity of the amino acid composition appears to be driven, at least inpart, via increase in fatty acid oxidation, reduction in levels of keycytokines and transcription pathways associated with liver inflammationand fibrosis.

Example 10 Hepatocyte Model for Steatosis and Inflammation

Hepatocyte lipotoxicity appears to be a central driver of hepaticcellular injury via oxidative stress and endoplasmic reticulum (ER)stress. The ability of amino acids to influence steatosis (lipidaccumulation) and inflammation in hepatocytes was assessed using humanprimary hepatocytes (Lonza, TRL).

Cell Seeding and Maintenance

Primary hepatocytes lot nos. from two healthy human donors were seededon day 0 at density of 6e04 cells in 96 well optical microplates(Thermofisher) in hepatocyte plating media (William's E medium (Gibco)supplemented with 10% heat-inactivated FBS (Atlanta Bio), 2 mM Glutamax(Gibco), 1×ITS plus (R&D systems), and 0.2% Primocin (InVivoGen) andincubated for 6 hours at 37° C., 5% CO₂. After 6 hours, cells werewashed twice with 150 ul William's E medium and incubated overnight at37° C., 5% CO₂ with serum-free hepatocytes culture media (Hepatocytesdefined medium (Corning)) supplemented with 5 ug human recombinant EGF(Corning), 2 mM Glutamax (Gibco), and 1× Penicillin/Streptomycin. On day1, cells were washed twice with 150 μL per well William's E medium(Gibco) and incubated for 24 h in the hepatocyte culture media in thesame conditions described above.

Amino Acids Pre-Treatment

On day 2, cells were washed twice with 150 ul DPBS 1× (Gibco) andmaintained in amino acid-free WEM (US Biologicals) containing a definedcustom amino acid concentration based on the mean physiologicalconcentrations in blood. The values are published in the HumanMetabolome Database (Wishart D S, Tzur D, Knox C, et al., HMDB: theHuman Metabolome Database. Nucleic Acids Res. 2007 January; 35 (Databaseissue):D521-6. 17202168; which is hereby incorporated by reference inits entirety). This custom media is supplemented with 11 mM Glucose,0.272 mM Sodium Pyruvate, and a dose curve of defined amino acidcompositions (i.e., vehicle, LIVRQ+N-acetylcysteine, LIVRQ,RQ+N-acetylcysteine, N-acetylcysteine alone, LIV, or individually withL-Leucine, L-Isoleucine, L-Valine, L-Arginine, L-Glutamine, andL-Cysteine) at various ranges of concentrations. Cells were maintainedin this defined media for 24 hours at 37° C., 5% CO₂.

Co-Treatment with Free Fatty Acids and Different Amino Acids Combination

After pre-treatment, cells were exposed to free fatty acids (FFA) at 250uM with a ratio of 2:1 (Oleate:Palmitate) supplemented with TNF-α(Thermofisher) at 1 ng/ml or vehicle. Cells were incubated with the FFAsmixture and the different amino acids combinations for 24 hours at 37°C., 5% CO₂. After 24 hours incubation, media was removed for cytokineanalysis and replaced by fresh media containing the same stimulusconditions and amino acid concentrations. Cells were incubated for anadditional 48 hours for a total of 72 hours of FFA and TNFα stimulation.

Cytokine Analysis after 24 h by ELISA

Human CCL2 (MCP-1) was measured by ELISA (Human CCK2/MCP-1 DuoSet ELISA,R&D Systems) at ⅕ or 1/10 dilution in 1× Reagent Diluent (ReagentAncillary Kit 2, R&D Systems). Data were normalized to the specific perwell cell density determined by nuclei count stained by Hoechst 3342(Life technologies) in the fluorescence microscopy described below.

Intracellular Lipid Accumulation Analysis after 72 h by FluorescenceMicroscopy

After 72 hours, cells were washed twice in 100 ul PBS 1× (Gibco), fixedwith 4% Paraformaldehyde, and washed twice with PBS 1× (100 ul). Afterfixation, lipids were stained with HCS LipidTOX Red Neutral(Thermofisher Scientific) diluted 1000× and nuclei were stained withHoechst 3342 (Life Technologies) diluted to 4 ug/ml. The LipidTOX™neutral lipid stain has an extremely high affinity for neutral lipiddroplets that was detected by fluorescence microscopy using a highcontent imager (Molecular Devices).

Results

Lipid Accumulation and Steatosis Phenotypes

Primary human hepatocytes from healthy donors were found to have lowlevels of lipid accumulation (FIG. 9A-9D). Treatment of the cells withfree fatty acids (FF)+TNFα induced lipid accumulation (FIG. 9I-9L) witha macro-steatosis phenotype. Treatment with LIVRQNAC changed thehepatocyte phenotypes from macro-steatosis to micro-steatosis (FIG.9E-9H).

MCP1/CCL2 Secretion

Tables 56-59 show the baseline subtracted secretion of MCP1/CCL2 inprimary human hepatocytes cells from two healthy donors (donor 1 forTables 56 and 57, and donor 2 for Tables 58 and 59). LIVRQNAC,LIVRQNAC+G, LIVRQNAC+S, LIVRQ and RQNAC significantly decreasedMCP1/CCL2 secretion in both donors. The combination LIV, however,significantly increased MCP1/CCL2 secretion only in one of the donors.The addition of arginine (R) and glutamine (Q) to a combination of LIVdecreased the secretion of MCP1/CCL2 in both donors compared to LIValone. Individually, N-acetyl cysteine and glutamine are shown tosignificantly decrease MCP1/CCL2 secretion, while arginine increasedMCP1 secretion. Isoleucine, Leucine and Valine did not have an effect onMCP1/CCL2 secretion.

TABLE 56 Changes in MCP1 expression for donor 1 upon administration ofamino acid compositions MCP1 expression relative to Control—Donor 1Number Amino Acid Conc. Std. of P- Supplement (X) Mean Deviation valuesvalue* Significance LIVRQNAC 40 −24.1616 0.032252 3 0.0001 **** LIVRQNAC30 −22.2916 2.119583 3 0.0001 **** LIVRQNAC 20 −18.4363 0.850597 30.0005 *** LIVRQNAC 10 −14.3383 1.854977 3 0.0074 ** LIVRQNAC 1 01.048045 3 LIVRQNAC + G 40 −22.0824 0.873105 3 0.0001 **** LIVRQNAC + G30 −19.2605 1.611788 3 0.0003 *** LIVRQNAC + G 20 −17.5807 2.893835 30.0009 *** LIVRQNAC + G 10 −13.7521 3.068991 3 0.0106 * LIVRQNAC + G 1 01.682719 3 LIVRQNAC + S 40 −32.4703 0.340537 3 0.0001 **** LIVRQNAC + S30 −30.768 1.339048 3 0.0001 **** LIVRQNAC + S 20 −25.5964 1.854519 30.0001 **** LIVRQNAC + S 10 −17.8326 1.974033 3 0.0008 *** LIVRQNAC + S1 2.37E−15 18.41384 3 LIV 40 15.52052 6.323205 3 0.0094 ** LIV 3012.3111 10.02706 3 0.0475 * LIV 20 12.6686 4.109608 3 0.0401 * LIV 10−5.18869 1.579468 3 0.6477 ns LIV 1 −1.2E−15 8.178943 3 LIVRQ 40−25.9576 0.484283 3 0.0028 ** LIVRQ 30 −23.6562 2.599721 3 0.0099 **LIVRQ 20 −13.4723 3.427666 3 0.6401 ns LIVRQ 10 −9.22141 7.599407 30.9986 ns LIVRQ 1 −8.23198 5.80889 3 RQNAC 40 −21.4681 2.903892 3 0.0003*** RQNAC 30 −17.1873 5.202568 3 0.0038 ** RQNAC 20 −12.1782 2.907484 30.0506 ns RQNAC 10 −8.89378 4.748653 3 0.206 ns RQNAC 1 1.18E−1510.02527 3 N-Acetyl Cysteine 40 −17.6065 1.211739 3 0.0009 *** N-AcetylCysteine 20 −10.8919 2.27818 3 0.0545 ns N-Acetyl Cysteine 10 −2.497558.795693 3 0.9424 ns N-Acetyl Cysteine 5 −0.76286 7.457085 3 0.9991 nsN-Acetyl Cysteine 0 0 6.716428 3

TABLE 57 Changes in MCP1 expression for donor 1 upon administration ofsingle amino acid compositions MCP1 expression relative to Control—Donor1 Number Amino Acid Conc. Std. of P- Supplement (μM) Mean Deviationvalues value* Significance Valine 23420 14.16805 19.23365 3 0.6777 nsValine 11710 77.73396 137.82 3 0.9998 ns Valine 4684 23.6867 46.48697 30.2502 ns Valine 234 −2.4E−15 13.86902 3 Arginine 5440 10.9386 4.79774 30.0057 ** Arginine 2720 6.526801 4.266971 3 0.1517 ns Arginine 10885.114414 4.685563 3 0.3321 ns Arginine 109 2.37E−15 0.666016 3 Glutamine22484 −21.8392 1.113443 3 0.0004 *** Glutamine 11242 −9.00139 1.68951 30.2459 ns Glutamine 3747 −0.89805 6.374471 3 0.9991 ns Glutamine 749 09.549143 3 Isoleucine 6639 −0.205 2.292188 3 0.9998 ns Isoleucine 3320−2.41722 2.382379 3 0.4907 ns Isoleucine 1328 −0.30729 2.409691 3 0.9992ns Isoleucine 66 −1.2E−15 3.163838 3 Leucine 15270 −1.36762 3.37035 30.8675 ns Leucine 7635 1.895506 3.757642 3 0.6872 ns Leucine 30543.340489 3.016641 3 0.2201 ns Leucine 153 5.92E−16 3.132507 3 N-AcetylCysteine 10000 −17.6065 1.211739 3 0.0009 *** N-Acetyl Cysteine 5000−10.8919 2.27818 3 0.0545 ns N-Acetyl Cysteine 2500 −2.49755 8.795693 30.9424 ns N-Acetyl Cysteine 1000 −0.76286 7.457085 3 0.9991 ns N-AcetylCysteine 0 0 6.716428 3

TABLE 58 Changes in MCP1 expression for donor 2 upon administration ofamino acid compositions MCP1 expression relative to Control—Donor 2Number Amino Acid Conc. Std. of P- Supplement (X) Mean Deviation valuesvalue* Significance LIVRQNAC 40 −24.5376 1.632923 3 0.0001 **** LIVRQNAC30 −13.6824 2.562571 3 0.0001 **** LIVRQNAC 20 −8.42053 1.545343 30.0001 **** LIVRQNAC 10 2.126223 0.453924 3 0.0007 *** LIVRQNAC 1−4.7E−15 0.412226 3 LIVRQNAC + G 40 −35.3651 2.08381 3 0.0007 ***LIVRQNAC + G 30 −30.3247 5.225183 3 0.001 *** LIVRQNAC + G 20 −17.07194.522244 3 0.0119 * LIVRQNAC + G 10 −14.2586 2.767898 3 0.049 *LIVRQNAC + G 1 −7.1E−15 7.613666 3 LIVRQNAC + S 40 −35.8381 1.404782 30.0001 **** LIVRQNAC + S 30 −30.9946 2.372062 3 0.0001 **** LIVRQNAC + S20 −16.8831 3.223007 3 0.0004 *** LIVRQNAC + S 10 −5.60595 10.2119 30.1887 LIVRQNAC + S 1 2.37E−15 4.4168 3 LIV 40 −46.7898 8.664441 30.3692 ns LIV 30 −34.5953 16.84743 3 0.6246 ns LIV 20 −28.0851 31.843483 0.7684 ns LIV 10 −11.0006 72.74556 3 0.9889 ns LIV 1 9.47E−15 60.936383 LIVRQ 40 −129.802 7.067989 3 0.0008 *** LIVRQ 30 −110.034 4.53852 30.0042 ** LIVRQ 20 −33.3611 31.87706 3 0.6524 LIVRQ 10 −3.30904 71.032673 0.9999 LIVRQ 1 −4.7E−15 46.12987 3 RQNAC 40 −133.48 1.908424 3 0.0006*** RQNAC 30 −123.712 1.043889 3 0.0013 ** RQNAC 20 −109.575 5.533323 30.0044 ** RQNAC 10 −55.8583 22.72309 3 0.2273 RQNAC 1 1.42E−14 43.790313 N-Acetyl 10000 −28.4419 1.694 3 0.0001 *** Cysteine N-Acetyl 5000−10.5725 4.362178 3 0.0012 ** Cysteine N-Acetyl 2500 −4.0591 5.600773 30.0572 ns Cysteine N-Acetyl 1000 1.602474 3.423109 3 0.0001 ****Cysteine N-Acetyl 0 0 2.068861 3 Cysteine

TABLE 59 Changes in MCP1 expression for donor 2 upon administration ofsingle amino acid compositions MCP1 expression relative to Control—Donor2 Number Amino Acid Conc. Std. of Supplement (μM) Mean Deviation valuesP-value* Significance Valine 23420 −30.7921 22.55378 3 0.6118 ns Valine11710 38.24762 28.44112 3 0.4268 ns Valine 4684 10.79011 51.87642 30.9835 ns Valine 234 −1.4E−14 30.91388 3 Arginine 5440 8.493664 22.983853 0.9913 ns Arginine 2720 24.06261 63.49489 3 0.7429 ns Arginine 108824.95224 52.94171 3 0.7192 ns Arginine 109 −4.7E−15 11.27976 3 Glutamine22484 −138.873 10.74317 3 0.0001 **** Glutamine 11242 −90.6558 15.439893 0.0037 ** Glutamine 3747 −45.0574 41.63249 3 0.2474 ns Glutamine 7492.84E−14 59.86955 3 0.7631 Isoleucine 6639 18.62132 26.01824 3 0.5663 nsIsoleucine 3320 −5.64461 7.719105 3 0.9882 ns Isoleucine 1328 26.623095.65413 3 0.2613 ns Isoleucine 66 0 4.245462 3 Leucine 15270 −26.643610.08177 3 0.2607 ns Leucine 7635 −2.98815 21.00205 3 0.9989 ns Leucine3054 16.11014 8.662188 3 0.68 ns Leucine 153 −4.7E−15 7.63396 3 N-Acetyl10000 −28.4419 1.694 3 0.0001 *** Cysteine N-Acetyl 5000 −10.57254.362178 3 0.0012 ** Cysteine N-Acetyl 2500 −4.0591 5.600773 3 0.0572 nsCysteine N-Acetyl 1000 1.602474 3.423109 3 0.0001 **** Cysteine N-Acetyl0 0 2.068861 3 Cysteine

Example 11 Hepatic Stellate Cell—TNFα Inflammatory Response

Methods

Primary human hepatic stellate cells were obtained from Samsara Sciencesbased on the following criteria for selecting donors: adult age (between18 and 50 years), normal BMI (>18.5 and <25), and absence of confoundingliver disease. Primary human hepatic stellate cells grown in CompleteHSC Medium to ˜80% confluence in T75 or T150 flasks below passage 10were seeded into sterile, collagen I coated, 96-well optical plasticmicroplates (ThermoScientific, 152036) at 4000 cells per well (˜1250cells per cm²) and incubated for 6 hours at 37° C., 5% CO₂ in ahumidified incubator.

After 6 hours, plates were removed from the incubator and the mediumgently pipetted off and washed once with 150 μL per well DPBS. The DPBSwas removed and the pretreatment medium (±single amino acid dropout,1×HMDB DMEM+3% dialyzed FBS+0.2% Primocin, ±supplemental amino aciddose; see experiment for medium composition) was applied to the cells at150 μL per well. Plates were returned to the incubator overnight, ˜14-15hours.

After overnight pretreatment, the medium was removed from the cells, andthe same pretreatment medium, now supplemented with 3 ng/mL TNFα isapplied. Each plate contained 3 ng/mL TNFα in 1× human plasma amino acid(HMDB or PAA) concentration medium, 0 ng/mL in 1×HMDB, and 3 ng/mLTNFα+50 nM Bengamide in 1×HMDB to serve as controls. Plates wereincubated for 12 hours at 37° C., 5% CO₂.

After 12 hour stimulus with TNFα, supernatant was removed and frozen at−80° C. in two separate aliquots. Plates were washed gently once withDPBS and 100 μL per well of 1×HMDB DMEM+3% dialyzed FBS+0.2%Primocin+10% CCK-8 viability reagent (Dojindo). Plates were incubatedfor 1 hour at 37° C., 5% CO₂.

After 1 hour of incubation, viability was measured on the Synergy platereader (Absorbance at 977 (test), 900 (reference), and 450 (CCK8) nm).Immediately, the medium was removed and the plates were fixed with 70 μLper well 4% paraformaldehyde in PBS at room temperature for 20 minutes,followed by two 150 μL PBS washes, and stored with 100 μL per well PBSat 4° C. until immunofluorescence staining.

Human CCL2/MCP1 and Human IL-6 were measured by ELISA (Human CCK2/MCP-1DuoSet ELISA, R&D Systems; Human IL-6 DuoSet ELISA, R&D Systems) at ⅕and 1/20 dilution in 1× Reagent Diluent (Reagent Ancillary Kit 2, R&DSystems). Data were normalized to the specific per well cell densitydetermined by Hoechst stained nuclei count.

Results

Pro-Inflammatory MCP-1 Chemokine Secretion

Tables 60-63 show per-cell normalized MCP-1 chemokine secretion inprimary human hepatic stellate cells from two donors as a fold changefrom the plasma amino acid background. Statistical significancecalculated by one-way ANOVA with Dunnett's multiple comparison testwithin each treatment group. LIVRQNAC+G and RQNAC singificantly decreaseMCP-1 secretion in both donors. LIVRQNAC, LIVRQNAC+S reduced MCP1secretion and was statistically significant in one of two donors.Individually, each of valine, arginine, and leucine had no significantimpact on MCP-1 secretion. Glutamine reduced MCP1 secretion in bothdonors but was only statistically significant in one of two donors.N-acetyl cysteine significantly reduced MCP-1 secretion in both donors.

TABLE 60 Changes in MCP1 secretion for donor 3 upon administration ofamino acid compositions Fold Change MCP1 Secretion Normalized Per CellStd. Number Amino Acid Conc. Devia- of Signif- P- Supplement (X) Meantion values icance value LIVRQNAC 40 0.6237 0.2500 3 ns 0.2763 LIVRQNAC30 0.6180 0.2436 3 ns 0.2657 LIVRQNAC 20 0.5679 0.1728 3 ns 0.1863LIVRQNAC 10 0.5548 0.2139 3 ns 0.1694 LIVRQNAC 1 1.0000 0.3619 3LIVRQNAC + G 40 0.6216 0.0903 3 ** 0.0036 LIVRQNAC + G 30 0.6742 0.05493 ** 0.0095 LIVRQNAC + G 20 0.6373 0.0888 3 ** 0.0047 LIVRQNAC + G 100.7075 0.0610 3 * 0.0179 LIVRQNAC + G 1 1.0000 0.1704 3 LIVRQNAC + S 400.5911 0.1451 3 ns 0.2045 LIVRQNAC + S 30 0.5932 0.1943 3 ns 0.2077LIVRQNAC + S 20 0.5760 0.1681 3 ns 0.1828 LIVRQNAC + S 10 0.6820 0.23963 ns 0.3845 LIVRQNAC + S 1 1.0000 0.4098 3 LIV 40 1.2677 0.5786 3 ns0.7802 LIV 30 1.3632 0.5837 3 ns 0.8368 LIV 20 1.3336 0.4754 3 ns 0.7964LIV 10 1.3745 0.5427 3 ns 0.9132 LIV 1 1.0000 0.3186 3 LIVRQ 40 1.30420.4140 3 ns 0.7695 LIVRQ 30 1.2208 0.4403 3 ns 0.9036 LIVRQ 20 0.99150.3521 3 ns 0.9999 LIVRQ 10 0.9968 0.3907 3 ns 0.9999 LIVRQ 1 1.00000.4257 3 RQNAC 40 0.3220 0.0282 3 **** 0.0001 RQNAC 30 0.4353 0.0941 3**** 0.0001 RQNAC 20 0.4629 0.0998 3 *** 0.0001 RQNAC 10 0.6513 0.0925 3** 0.0028 RQNAC 1 1.0000 0.1132 3 N-Acetyl Cysteine 40 0.4485 0.0587 3*** 0.0002 N-Acetyl Cysteine 20 0.5413 0.1018 3 *** 0.0009 N-AcetylCysteine 10 0.6565 0.0502 3 ** 0.007  N-Acetyl Cysteine 5 0.8492 0.15153 ns 0.2738 N-Acetyl Cysteine 0 1.0000 0.1142 3

TABLE 61 Changes in MCP1 secretion for donor 3 upon administration ofsingle amino acid compositions Fold Change MCP1 Secretion Normalized PerCell Std. Number Amino Acid Conc. Devia- of Signif- P- Supplement (μM)Mean tion values icance value Valine 23420 1.2651 0.1295 3 ns 0.1126Valine 11710 1.0204 0.1126 3 ns 0.9956 Valine 4684 1.0630 0.0878 3 ns0.8999 Valine 234 1.0000 0.2008 3 Arginine 5440 0.7840 0.2753 3 ns0.7069 Arginine 2720 0.8821 0.2249 3 ns 0.9264 Arginine 1088 0.94350.3221 3 ns 0.9903 Arginine 109 1.0000 0.3404 3 Glutamine 22484 0.62120.1952 3 ns 0.2465 Glutamine 11242 0.6106 0.2085 3 ns 0.226  Glutamine3747 0.6036 0.2596 3 ns 0.2135 Glutamine 749 0.7048 0.2473 3 ns 0.4593Glutamine 562 1.0000 0.2185 3 Isoleucine 6639 1.2084 0.1334 3 ns 0.284 Isoleucine 3320 1.2169 0.0589 3 ns 0.2565 Isoleucine 1328 1.5550 0.20703 ** 0.0038 Isoleucine 66 1.0000 0.1188 3 Leucine 15270 1.1808 0.2601 3ns 0.5156 Leucine 7635 1.3054 0.1748 3 ns 0.1491 Leucine 3054 1.14790.0605 3 ns 0.6605 Leucine 153 1.0000 0.0784 3 N-Acetyl Cysteine 100000.4485 0.0587 3 *** 0.0002 N-Acetyl Cysteine 5000 0.5413 0.1018 3 ***0.0009 N-Acetyl Cysteine 2500 0.6565 0.0502 3 ** 0.007  N-AcetylCysteine 1000 0.8492 0.1515 3 ns 0.2738 N-Acetyl Cysteine 0 1.00000.1142 3

TABLE 62 Changes in MCP1 secretion for donor 4 upon administration ofamino acid compositions Fold Change MCP1 Secretion Normalized Per CellStd. Number Amino Acid Conc. Devia- of Signif- P- Supplement (X) Meantion values icance value LIVRQNAC 40 0.7791 0.0740 3 ns 0.1328 LIVRQNAC30 0.6333 0.1114 3 * 0.0116 LIVRQNAC 20 0.6997 0.1013 3 * 0.0352LIVRQNAC 10 0.8114 0.1271 3 ns 0.2216 LIVRQNAC 1 1.0000 0.1607 3LIVRQNAC + G 40 0.6738 0.0979 3 * 0.0454 LIVRQNAC + G 30 0.7117 0.0783 3ns 0.0794 LIVRQNAC + G 20 0.6735 0.1127 3 * 0.0452 LIVRQNAC + G 100.7682 0.0563 3 ns 0.1778 LIVRQNAC + G 1 1.0000 0.2452 3 LIVRQNAC + S 400.5780 0.0781 3 ** 0.0025 LIVRQNAC + S 30 0.5393 0.1185 3 ** 0.0013LIVRQNAC + S 20 0.6487 0.0732 3 ** 0.0085 LIVRQNAC + S 10 0.6872 0.01183 * 0.017  LIVRQNAC + S 1 1.0000 0.1803 3 LIV 40 0.7010 0.1399 3 **0.0059 LIV 30 0.8883 0.0530 3 ns 0.3745 LIV 20 0.9284 0.0579 3 ns 0.7114LIV 10 0.8663 0.0569 3 ns 0.2428 LIV 1 1.0000 0.0928 3 LIVRQ 40 1.22350.0592 3 ns 0.4365 LIVRQ 30 1.1653 0.0558 3 ns 0.6679 LIVRQ 20 0.88450.2698 3 ns 0.862  LIVRQ 10 1.0110 0.0738 3 ns 0.9999 LIVRQ 1 1.00000.3016 3 RQNAC 40 0.4312 0.0994 3 *** 0.0006 RQNAC 30 0.3910 0.0649 3*** 0.0003 RQNAC 20 0.5579 0.2079 3 ** 0.0037 RQNAC 10 0.5545 0.0663 3** 0.0035 RQNAC 1 1.0000 0.0987 3 N-Acetyl Cysteine 40 0.5011 0.0756 3*** 0.0001 N-Acetyl Cysteine 20 0.6728 0.1024 3 ** 0.003  N-AcetylCysteine 10 0.8033 0.1101 3 ns 0.058  N-Acetyl Cysteine 5 0.6437 0.06483 ** 0.0017 N-Acetyl Cysteine 0 1.0000 0.0673 3

TABLE 63 Changes in MCP1 secretion for donor 4 upon administration ofsingle amino acid compositions Fold Change MCP1 Secretion Normalized PerCell Std. Number Amino Acid Conc. Devia- of Signif- P- Supplement (μM)Mean tion values icance value Valine 23420 1.1525 0.0406 3 ns 0.9999Valine 11710 1.1544 0.1743 3 ns 0.8877 Valine 4684 1.0942 0.0846 3 ns0.3545 Valine 234 1.0000 0.1464 3 Arginine 5440 0.9456 0.0639 3 ns0.9076 Arginine 2720 1.0446 0.0741 3 ns 0.9449 Arginine 1088 1.04530.1733 3 ns 0.9423 Arginine 109 1.0000 0.1486 3 Glutamine 22484 0.70390.0544 3 ** 0.0065 Glutamine 11242 0.7129 0.2237 3 ** 0.0077 Glutamine3747 0.6639 0.0467 3 ** 0.0027 Glutamine 749 0.7782 0.0860 3 * 0.0452Glutamine 562 1.0000 0.0709 6 Isoleucine 6639 0.9103 0.0536 3 ns 0.5597Isoleucine 3320 0.8830 0.0872 3 ns 0.3538 Isoleucine 1328 1.3338 0.10993 ** 0.0044 Isoleucine 66 1.0000 0.0853 3 Leucine 15270 1.5745 0.0844 3ns 0.1886 Leucine 7635 1.7129 0.6026 3 ns 0.0885 Leucine 3054 1.53420.1746 3 ns 0.2332 Leucine 153 1.0000 0.2040 3 N-Acetyl Cysteine 100000.5011 0.0756 3 *** 0.0001 N-Acetyl Cysteine 5000 0.6728 0.1024 3 **0.003  N-Acetyl Cysteine 2500 0.8033 0.1101 3 ns 0.058  N-AcetylCysteine 1000 0.6437 0.0648 3 ** 0.0017 N-Acetyl Cysteine 0 1.00000.0673 3IL-6 Cytokine Secretion

Tables 64-67 show per-cell normalized IL-6 cytokine secretion in primaryhuman hepatic stellate cells from two donors as a fold change from theplasma amino acid background. Statistical significance calculated byone-way ANOVA with Dunnett's multiple comparison test within eachtreatment group. LIVRQNAC, LIVRQNAC+S and RQNAC significantly reducedIL-6 secretion in one of two donors. LIVRQNAC+G, LIVRQNAC+S and RQNACdecreased IL-6 secretion in both donors. LIV and LIVRQ did not have asignificant impact on IL-6 secretion in either donor. Individually,valine, arginine, isoleucine, and leucine had no significant effect onIL-6 secretion. N-acetyl cysteine reduced IL-6 secretion in both donorsbut was only statistically significant in one of two donors. Glutaminesignificantly reduced IL-6 secretion in both donors.

TABLE 64 Changes in IL-6 cytokine secretion for donor 1 uponadministration of amino acid compositions Fold Change IL-6 SecretionNormalized Per Cell Std. Number Amino Acid Conc. Devia- of Signif- P-Supplement (X) Mean tion values icance value LIVRQNAC 40 0.4857 0.0915 3*** 0.0004 LIVRQNAC 30 0.5667 0.0941 3 ** 0.0014 LIVRQNAC 20 0.66710.0431 3 ** 0.0088 LIVRQNAC 10 0.6579 0.1231 3 ** 0.0074 LIVRQNAC 11.0000 0.1361 3 LIVRQNAC + G 40 0.4995 0.1427 3 ns 0.0949 LIVRQNAC + G30 0.5722 0.2185 3 ns 0.1679 LIVRQNAC + G 20 0.6185 0.1769 3 ns 0.2376LIVRQNAC + G 10 0.7040 0.2809 3 ns 0.4276 LIVRQNAC + G 1 1.0000 0.3513 3LIVRQNAC + S 40 0.5397 0.1569 3 * 0.0105 LIVRQNAC + S 30 0.5513 0.11903 * 0.0122 LIVRQNAC + S 20 0.6264 0.1593 3 * 0.0338 LIVRQNAC + S 100.6799 0.1218 3 ns 0.0703 LIVRQNAC + S 1 1.0000 0.1671 3 LIV 40 1.35360.4767 3 ns 0.6216 LIV 30 1.2423 0.3135 3 ns 0.8437 LIV 20 1.2321 0.48183 ns 0.8611 LIV 10 1.1421 0.3489 3 ns 0.9704 LIV 1 1.0000 0.1647 3 LIVRQ40 0.8274 0.2003 3 ns 0.7863 LIVRQ 30 0.8880 0.2175 3 ns 0.938  LIVRQ 200.8468 0.1100 3 ns 0.8431 LIVRQ 10 0.9247 0.2696 3 ns 0.984  LIVRQ 11.0000 0.3311 3 RQNAC 40 0.3958 0.0947 3 * 0.0109 RQNAC 30 0.4433 0.13173 * 0.0177 RQNAC 20 0.4936 0.1079 3 * 0.0297 RQNAC 10 0.5729 0.1741 3 ns0.0674 RQNAC 1 1.0000 0.3440 3 N-Acetyl Cysteine 40 0.5716 0.2306 3 ns0.2067 N-Acetyl Cysteine 20 0.6121 0.1718 3 ns 0.2729 N-Acetyl Cysteine10 0.7354 0.2816 3 ns 0.5703 N-Acetyl Cysteine 5 0.7141 0.2509 3 ns0.5098 N-Acetyl Cysteine 0 1.0000 0.3472 3

TABLE 65 Changes in IL-6 cytokine secretion for donor 1 uponadministration of single amino acid compositions Fold Change IL-6Secretion Normalized Per Cell Std. Number Amino Acid Conc. Devia- ofSignif- P- Supplement (μM) Mean tion values icance value Valine 234201.0404 0.2175 3 ns 0.9949 Valine 11710 0.9562 0.3332 3 ns 0.9935 Valine4684 0.9790 0.1777 3 ns 0.9993 Valine 234 1.0000 0.2868 3 Arginine 54400.7776 0.1994 3 ns 0.6927 Arginine 2720 1.0231 0.4381 3 ns 0.9993Arginine 1088 0.9828 0.2957 3 ns 0.9997 Arginine 109 1.0000 0.1728 3Glutamine 22484 0.5138 0.0818 3 ** 0.0046 Glutamine 11242 0.5136 0.11893 ** 0.0046 Glutamine 3747 0.5460 0.0891 3 ** 0.0072 Glutamine 7490.6320 0.1181 3 * 0.0249 Glutamine 562 1.0000 0.2226 3 Isoleucine 66391.0859 0.1489 3 ns 0.764  Isoleucine 3320 1.1156 0.0776 3 ns 0.5903Isoleucine 1328 1.0233 0.1536 3 ns 0.9922 Isoleucine 66 1.0000 0.1276 3Leucine 15270 1.0767 0.0246 3 ns 0.853  Leucine 7635 1.1215 0.0872 3 ns0.6249 Leucine 3054 1.1762 0.2273 3 ns 0.3655 Leucine 153 1.0000 0.15353 N-Acetyl Cysteine 10000 0.5716 0.2306 3 ns 0.2067 N-Acetyl Cysteine5000 0.6121 0.1718 3 ns 0.2729 N-Acetyl Cysteine 2500 0.7354 0.2816 3 ns0.5703 N-Acetyl Cysteine 1000 0.7141 0.2509 3 ns 0.5098 N-AcetylCysteine 0 1.0000 0.3472 3

TABLE 66 Changes in IL-6 cytokine secretion for donor 2 uponadministration of amino acid compositions Fold Change IL-6 SecretionNormalized Per Cell Std. Number Amino Acid Conc. Devia- of Signif- P-Supplement (X) Mean tion values icance value LIVRQNAC 40 0.9911 0.1150 3ns 0.9998 LIVRQNAC 30 0.9560 0.0473 3 ns 0.9404 LIVRQNAC 20 1.00080.1450 3 ns 0.9999 LIVRQNAC 10 1.0845 0.0707 3 ns 0.6567 LIVRQNAC 11.0000 0.0553 3 LIVRQNAC + G 40 0.8055 0.1705 3 ns 0.4153 LIVRQNAC + G30 0.8218 0.1567 3 ns 0.4855 LIVRQNAC + G 20 0.9236 0.1642 3 ns 0.9342LIVRQNAC + G 10 1.1076 0.2097 3 ns 0.8216 LIVRQNAC + G 1 1.0000 0.0416 3LIVRQNAC + S 40 0.9508 0.0933 3 ns 0.967  LIVRQNAC + S 30 0.8581 0.03643 ns 0.4836 LIVRQNAC + S 20 0.8289 0.0765 3 ns 0.3356 LIVRQNAC + S 100.8487 0.1018 3 ns 0.432  LIVRQNAC + S 1 1.0000 0.2312 3 LIV 40 0.91220.0773 3 ns 0.8233 LIV 30 1.0994 0.0987 3 ns 0.7586 LIV 20 1.0400 0.23303 ns 0.9857 LIV 10 0.9579 0.1077 3 ns 0.9828 LIV 1 1.0000 0.0540 3 LIVRQ40 0.9327 0.0639 3 ns 0.8313 LIVRQ 30 0.8421 0.1125 3 ns 0.2361 LIVRQ 200.7871 0.0932 3 ns 0.0841 LIVRQ 10 0.8693 0.0750 3 ns 0.3744 LIVRQ 11.0000 0.1428 3 RQNAC 40 0.8711 0.0816 3 ns 0.5267 RQNAC 30 0.74600.1133 3 ns 0.0843 RQNAC 20 0.7838 0.0708 3 ns 0.1544 RQNAC 10 0.87810.1566 3 ns 0.5705 RQNAC 1 1.0000 0.1557 3 N-Acetyl Cysteine 40 0.70640.0418 3 ns 0.0508 N-Acetyl Cysteine 20 0.8111 0.1049 3 ns 0.2549N-Acetyl Cysteine 10 0.9180 0.2230 3 ns 0.8353 N-Acetyl Cysteine 50.9161 0.1067 3 ns 0.8252 N-Acetyl Cysteine 0 1.0000 0.0632 3

TABLE 67 Changes in IL-6 cytokine secretion for donor 2 uponadministration of single amino acid compositions Fold Change IL-6Secretion Normalized Per Cell Std. Number Amino Acid Conc. Devia- ofSignif- P- Supplement (μM) Mean tion values icance value Valine 234200.9015 0.0930 3 ns 0.4967 Valine 11710 0.9218 0.1179 3 ns 0.6516 Valine4684 1.0383 0.1014 3 ns 0.9291 Valine 234 1.0000 0.0696 3 Arginine 54400.8895 0.0897 3 ns 0.547  Arginine 2720 0.9401 0.1611 3 ns 0.8654Arginine 1088 0.9924 0.0692 3 ns 0.9996 Arginine 109 1.0000 0.1263 3Glutamine 22484 0.5993 0.0611 3 **** 0.0001 Glutamine 11242 0.64780.0371 3 **** 0.0001 Glutamine 3747 0.7100 0.0356 3 *** 0.0003 Glutamine749 0.7673 0.0222 3 ** 0.0017 Glutamine 562 1.0000 0.1027 6 Isoleucine6639 1.1648 0.1125 3 ns 0.1448 Isoleucine 3320 0.9096 0.0916 3 ns 0.5304Isoleucine 1328 1.1020 0.0987 3 ns 0.4446 Isoleucine 66 1.0000 0.0641 3Leucine 15270 1.0183 0.1155 3 ns 0.9795 Leucine 7635 0.9574 0.0590 3 ns0.8187 Leucine 3054 1.0011 0.0618 3 ns 0.9999 Leucine 153 1.0000 0.02773 N-Acetyl Cysteine 10000 0.7064 0.0418 3 ns 0.0508 N-Acetyl Cysteine5000 0.8111 0.1049 3 ns 0.2549 N-Acetyl Cysteine 2500 0.9180 0.2230 3 ns0.8353 N-Acetyl Cysteine 1000 0.9161 0.1067 3 ns 0.8252 N-AcetylCysteine 0 1.0000 0.0632 3

Example 12 TGFβ1 Fibrogenic Gene Expression of Hepatic Stellate Cell

Primary human hepatic stellate cells were obtained from Samsara Sciencesbased on the following criteria for selecting donors: adult age (between18 and 50 years), normal BMI (>18.5 and <25), and absence of confoundingliver disease. Cells grown in Complete HSC Medium to ˜80% confluence inT75 or T150 flasks below passage 10 were seeded into sterile, collagen Icoated, 96-well optical plastic microplates (ThermoScientific, 152036)at 6000 cells per well (˜1250 cells per cm2) and incubated overnight at37° C., 5% CO₂ in a humidified incubator in DMEM with 2% Fetal BovineSerum and 1% Antibiotic-Antimycotic.

After the overnight incubation, plates were removed from the incubatorand the medium was gently pipetted off and washed twice with 150 μL perwell DPBS. The DPBS was removed and the pretreatment medium (±singleamino acid dropout, 1×HMDB DMEM+1% Antibiotic-Antimycotic, 10 mM HEPES,±supplemental amino acid dose; see experiment for medium composition)was applied to the cells at 150 μL per well. Plates were returned to theincubator for 10.5 hours.

After 10.5 hour pretreatment, the medium was removed from the cells, andthe same pretreatment medium, now supplemented with 3 ng/mL TGFβ1, wasapplied. Each plate contained 3 ng/mL TGFβ1 in 1× human plasma aminoacid (HMDB or PAA) concentration medium, 0 ng/mL in 1×HMDB, and 3 ng/mLTGFβ1+20 μM Silybin in 1×HMDB to serve as controls. Plates were thenincubated for 24 hours at 37° C., 5% CO₂.

After 24 hour stimulus, supernatant was removed and frozen at −80° C. intwo separate aliquots. The cells were then washed with 125 μL per wellBuffer FCW (FastLane Cell Multiplex NR Kit, Qiagen, 216713). The washbuffer was immediately removed and 50 μL of Cell Processing Mix(containing genomic DNA Wipeout buffer) was applied to lyse cells,incubating for 10 minutes at room temperature. RNA lysate was thentransferred to 96-well qPCR plates, sealed, and gDNA was digested onthermal cycler at 75° C. for 5 minutes. RNA lysate was frozen at −80° C.

Each 20 μL one-step RT-qPCR reaction contained 4 μL of RNA lysate. Geneexpression of Col1a1, Timp2, and Gapdh were multiplexed using the HEX,Cy5, and FAM fluorescent channels, respectively, with commerciallyavailable primer-probe mixes (the Human Col1a1 Primer-Probe Set, HEX;the Human Timp2 Primer-Probe Set, Cy5; and the Human Gapdh Primer-ProbeSet, FAM from IDT). Gene expression was evaluated using the ΔΔCq methodwithin each single amino acid dropout and supplementation by normalizingto its own 1×HMDB concentration.

Human Procollagen Iα1 was measured from the supernatant by ELISA (HumanPro-Collagen I alpha 1 DuoSet ELISA, R&D Systems) at 1/100 dilution in1× Reagent Diluent (Reagent Ancillary Kit 2, R&D Systems).

Results

Col1a1 Gene Expression

Tables 68, 69, 69-1, 69-2, 69-3, and 69-4 show the mean fold change inCol1a1 gene expression in primary human hepatic stellate cells fromthree different healthy donors. LIVRQNAC and LIVRQNAC+S showedsignificantly decreased Col1a1 gene expression in two of three donors.LIVRQNAC+G and RQNAC showed significantly decreased Col1a1 expression inall three donors. LIVRQ showed a significant change in Col1a1 geneexpression in only one donor. LIV alone did not significantly changeCol1a1 gene expression.

Each of leucine, isoleucine, valine, and arginine did not significantlychange Col1a1 gene expression in any donor when the amino acid wasadministered alone. Glutamine decreased Col1a1 gene expression in two ofthree donors. N-acetyl cysteine significantly reduced Col1a1 geneexpression in all three donors.

TABLE 68 Fold change of Col1a1 gene expression after administration ofan amino acid composition, normalized to Gapdh expression in a firstdonor Col1a1 Fold Expression Relative to Control Num- Std. ber AminoAcid Conc. Devia- of P- Signif- Supplement (X) Mean tion values value*icance LIVRQNAC 40 0.91 0.08 4 ns 0.401  LIVRQNAC 30 0.87 0.10 4 ns0.1073 LIVRQNAC 20 0.88 0.04 4 ns 0.1483 LIVRQNAC 10 0.90 0.08 4 ns0.3035 LIVRQNAC 1 1.00 0.10 4 LIVRQNAC + G 40 0.73 0.15 4 ** 0.0053LIVRQNAC + G 30 0.79 0.08 4 * 0.0252 LIVRQNAC + G 20 0.84 0.08 4 ns0.1181 LIVRQNAC + G 10 0.79 0.11 4 * 0.0286 LIVRQNAC + G 1 1.00 0.03 4LIVRQNAC + S 40 0.79 0.05 4 * 0.0325 LIVRQNAC + S 30 0.86 0.13 4 ns0.1848 LIVRQNAC + S 20 0.96 0.10 4 ns 0.9287 LIVRQNAC + S 10 0.85 0.12 4ns 0.1566 LIVRQNAC + S 1 1.00 0.10 4 LIV 40 0.93 0.03 4 ns 0.5561 LIV 301.04 0.07 4 ns 0.8872 LIV 20 1.04 0.09 4 ns 0.9069 LIV 10 1.05 0.10 4 ns0.8156 LIV 1 1.00 0.07 4 LIVRQ 40 0.75 0.03 4 *** 0.001  LIVRQ 30 0.730.05 4 *** 0.0004 LIVRQ 20 0.80 0.03 4 ** 0.0054 LIVRQ 10 0.84 0.08 4 *0.0208 LIVRQ 1 1.01 0.13 4 RQNAC 40 0.51 0.07 4 **** 0.0001 RQNAC 300.49 0.02 4 **** 0.0001 RQNAC 20 0.59 0.04 4 **** 0.0001 RQNAC 10 0.680.07 4 **** 0.0001 RQNAC 1 1.00 0.11 4 N-Acetyl Cysteine 40 0.76 0.06 4** 0.0011 N-Acetyl Cysteine 20 1.02 0.08 4 ns 0.9921 N-Acetyl Cysteine10 1.07 0.08 4 ns 0.5517 N-Acetyl Cysteine 5 1.00 0.08 4 ns 0.9999N-Acetyl Cysteine 0 1.00 0.06 4

TABLE 69 Fold change of Col1a1 gene expression after administration of asingle amino acid composition, normalized to Gapdh expression in thefirst donor Col1a1 Fold Expression Relative to Control Std. Number AminoAcid Conc. Devia- of P- Signif- Supplement (μM) Mean tion values value*icance Valine 23420 1.00 0.05 4 ns 0.9996 Valine 11710 1.09 0.17 4 ns0.5528 Valine 4684 1.05 0.11 4 ns 0.8851 Valine 234 1.00 0.08 4 Arginine5440 1.12 0.18 4 ns 0.2151 Arginine 2720 1.03 0.03 4 ns 0.9625 Arginine1088 0.99 0.06 4 ns 0.9989 Arginine 109 1.00 0.03 4 Glutamine 22484 0.530.01 4 **** 0.0001 Glutamine 11242 0.62 0.05 4 **** 0.0001 Glutamine3747 0.70 0.03 3 **** 0.0001 Glutamine 749 1.00 0.07 4 ns 0.9999Glutamine 562 1.00 0.07 3 Isoleucine 6639 1.11 0.07 4 ns 0.7553Isoleucine 3320 1.10 0.14 4 ns 0.7944 Isoleucine 1328 1.05 0.22 4 ns0.9831 Isoleucine 66 1.01 0.21 4 Leucine 15270 0.99 0.10 4 ns 0.994 Leucine 7635 1.12 0.16 4 ns 0.5049 Leucine 3054 1.11 0.15 4 ns 0.5499Leucine 153 1.00 0.11 4 N-Acetyl Cysteine 10000 0.76 0.06 4 ** 0.0011N-Acetyl Cysteine 5000 1.02 0.08 4 ns 0.9921 N-Acetyl Cysteine 2500 1.070.08 4 ns 0.5517 N-Acetyl Cysteine 1000 1.00 0.08 4 ns 0.9999 N-AcetylCysteine 0 1.00 0.06 4

TABLE 69-1 Fold change of Col1a1 gene expression after administration ofan amino acid composition, normalized to Gapdh expression in seconddonor. Col1a1 Fold Expression Relative to Control Num- Std. ber AminoAcid Conc. Devia- of P- Signif- Supplement (X) Mean tion values value*icance LIVRQNAC 40 0.72 0.05 4 **** 0.0001 LIVRQNAC 30 0.72 0.02 4 ****0.0001 LIVRQNAC 20 0.70 0.03 4 **** 0.0001 LIVRQNAC 10 0.71 0.08 4 ****0.0001 LIVRQNAC 1 1.00 0.02 4 LIVRQNAC + G 40 0.60 0.09 4 **** 0.0001LIVRQNAC + G 30 0.68 0.07 4 *** 0.0001 LIVRQNAC + G 20 0.71 0.09 4 ***0.0003 LIVRQNAC + G 10 0.69 0.06 4 *** 0.0002 LIVRQNAC + G 1 1.00 0.07 4LIVRQNAC + S 40 0.66 0.02 4 **** 0.0001 LIVRQNAC + S 30 0.69 0.06 4 ****0.0001 LIVRQNAC + S 20 0.76 0.05 4 *** 0.0002 LIVRQNAC + S 10 0.77 0.044 *** 0.0003 LIVRQNAC + S 1 1.00 0.11 4 LIV 40 1.20 0.21 4 ns 0.1032 LIV30 1.10 0.09 4 ns 0.6074 LIV 20 1.10 0.04 4 ns 0.6031 LIV 10 1.02 0.08 4ns 0.9981 LIV 1 1.00 0.11 4 LIVRQ 40 1.23 0.13 4 ns 0.1945 LIVRQ 30 1.120.13 4 ns 0.7176 LIVRQ 20 1.08 0.24 4 ns 0.8874 LIVRQ 10 1.14 0.16 4 ns0.5632 LIVRQ 1 1.00 0.11 4 RQNAC 40 0.54 0.03 4 **** 0.0001 RQNAC 300.55 0.06 4 **** 0.0001 RQNAC 20 0.58 0.04 4 **** 0.0001 RQNAC 10 0.730.04 4 *** 0.0007 RQNAC 1 1.01 0.16 4 N-Acetyl Cysteine 40 0.57 0.06 4**** 0.0001 N-Acetyl Cysteine 20 0.69 0.06 4 **** 0.0001 N-AcetylCysteine 10 0.69 0.09 4 *** 0.0001 N-Acetyl Cysteine 5 0.69 0.05 4 ***0.0001 N-Acetyl Cysteine 0 1.00 0.10 4

TABLE 69-2 Fold change of Col1a1 gene expression after administration ofa single amino acid composition, normalized to Gapdh expression insecond donor. Col1a1 Fold Expression Relative to Control Std. NumberAmino Acid Conc. Devia- of P- Signif- Supplement (μM) Mean tion valuesvalue* icance Valine 23420 1.05 0.03 4 ns 0.9194 Valine 11710 0.98 0.114 ns 0.9827 Valine 4684 1.05 0.18 4 ns 0.8893 Valine 234 1.00 0.11 4Arginine 5440 1.15 0.10 4 ns 0.2773 Arginine 2720 1.15 0.14 4 ns 0.2759Arginine 1088 0.99 0.15 4 ns 0.9938 Arginine 109 1.00 0.12 4 Glutamine22484 0.86 0.07 4 ns 0.1411 Glutamine 11242 0.91 0.09 4 ns 0.4365Glutamine 3747 1.04 0.14 4 ns 0.9811 Glutamine 749 1.02 0.13 4 ns 0.9988Glutamine 562 1.01 0.12 8 Isoleucine 6639 1.03 0.07 4 ns 0.8931Isoleucine 3320 0.99 0.08 4 ns 0.9841 Isoleucine 1328 0.97 0.10 4 ns0.9157 Isoleucine 66 1.00 0.02 4 Leucine 15270 1.13 0.14 4 ns 0.0811Leucine 7635 1.05 0.05 4 ns 0.7277 Leucine 3054 1.06 0.03 4 ns 0.5342Leucine 153 1.00 0.03 4 N-Acetyl Cysteine 10000 0.57 0.06 4 **** 0.0001N-Acetyl Cysteine 5000 0.69 0.06 4 **** 0.0001 N-Acetyl Cysteine 25000.69 0.09 4 *** 0.0001 N-Acetyl Cysteine 1000 0.69 0.05 4 *** 0.0001N-Acetyl Cysteine 0 1.00 0.10 4

TABLE 69-3 Fold change of Col1a1 gene expression after administration ofan amino acid composition, normalized to Gapdh expression in thirddonor. Col1a1 Fold Expression Relative to Control Std. Number Amino AcidConc. Devia- of P- Signif- Supplement (X) Mean tion values value* icanceLIVRQNAC 40 0.81 0.09 4 ** 0.008  LIVRQNAC 30 0.70 0.06 4 *** 0.0001LIVRQNAC 20 0.79 0.08 4 ** 0.0035 LIVRQNAC 10 0.79 0.07 4 ** 0.0039LIVRQNAC 1 1.00 0.06 4 LIVRQNAC + G 40 0.63 0.10 4 *** 0.0002 LIVRQNAC +G 30 0.64 0.02 4 *** 0.0003 LIVRQNAC + G 20 0.75 0.14 4 ** 0.005 LIVRQNAC + G 10 0.71 0.11 4 ** 0.0017 LIVRQNAC + G 1 1.00 0.03 4LIVRQNAC + S 40 0.79 0.11 4 * 0.0316 LIVRQNAC + S 30 0.79 0.04 4 *0.0309 LIVRQNAC + S 20 0.77 0.09 4 * 0.0208 LIVRQNAC + S 10 0.85 0.09 4ns 0.1434 LIVRQNAC + S 1 1.01 0.16 4 LIV 40 1.00 0.16 4 ns 0.9999 LIV 300.94 0.16 4 ns 0.8685 LIV 20 1.08 0.08 4 ns 0.6767 LIV 10 0.93 0.04 4 ns0.7713 LIV 1 1.00 0.05 4 LIVRQ 40 1.00 0.05 4 ns 0.9999 LIVRQ 30 1.070.13 4 ns 0.8753 LIVRQ 20 1.10 0.13 4 ns 0.6983 LIVRQ 10 1.05 0.21 4 ns0.9641 LIVRQ 1 1.00 0.07 4 RQNAC 40 0.64 0.05 4 *** 0.0003 RQNAC 30 0.700.13 4 ** 0.0018 RQNAC 20 0.66 0.05 4 *** 0.0005 RQNAC 10 0.87 0.15 4 ns0.2175 RQNAC 1 1.00 0.04 4 N-Acetyl Cysteine 40 0.62 0.01 4 *** 0.0005N-Acetyl Cysteine 20 0.73 0.10 4 ** 0.0083 N-Acetyl Cysteine 10 0.820.09 4 ns 0.0909 N-Acetyl Cysteine 5 0.91 0.12 4 ns 0.4954 N-AcetylCysteine 0 1.01 0.16 4

TABLE 69-4 Fold change of Col1a1 gene expression after administration ofa single amino acid composition, normalized to Gapdh expression insecond donor. Col1a1 Fold Expression Relative to Control Std. NumberAmino Acid Conc. Devia- of P- Signif- Supplement (μM) Mean tion valuesvalue* icance Valine 23420 1.13 0.12 4 ns 0.7199 Valine 11710 1.27 0.314 ns 0.1735 Valine 4684 1.22 0.16 4 ns 0.3247 Valine 234 1.01 0.13 4Arginine 5440 1.02 0.09 4 ns 0.9702 Arginine 2720 0.99 0.09 4 ns 0.9973Arginine 1088 0.95 0.02 4 ns 0.5384 Arginine 109 1.00 0.05 4 Glutamine22484 0.81 0.11 4 * 0.0113 Glutamine 11242 0.81 0.11 4 ** 0.0087Glutamine 3747 1.00 0.03 4 ns 0.9999 Glutamine 749 0.96 0.07 4 ns 0.8697Glutamine 562 1.00 0.10 8 Isoleucine 6639 1.03 0.04 4 ns 0.9974Isoleucine 3320 0.94 0.13 4 ns 0.8329 Isoleucine 1328 0.94 0.17 4 ns0.7947 Isoleucine 66 1.02 0.20 4 Leucine 15270 1.07 0.12 4 ns 0.9535Leucine 7635 1.00 0.16 4 ns 0.998 Leucine 3054 1.08 0.23 4 ns 0.9185Leucine 153 1.01 0.19 4 N-Acetyl Cysteine 10000 0.62 0.01 4 *** 0.0005N-Acetyl Cysteine 5000 0.73 0.10 4 ** 0.0083 N-Acetyl Cysteine 2500 0.820.09 4 ns 0.0909 N-Acetyl Cysteine 1000 0.91 0.12 4 ns 0.4954 N-AcetylCysteine 0 1.01 0.16 4

Procollagen Iα1 Secretion

Tables 70, 71, 71-1, 71-2, 71-3, and 71-4 show the fold change inprocollagen Iα1 in primary human hepatic stellate cells from threedifferent healthy donors normalized to their respective baseline aminoacid conditions. Statistical significance calculated by one-way ANOVAwith Dunnett's multiple comparison test within each treatment group. Thecombination LIV significantly increased procollagen Iα1 secretion in allthree donors. The addition of arginine (R) and glutamine (Q) to acombination of LIV counteracted the profibrogenic effect of LIV alone.LIVRQNAC, LIVRQNAC+G, LIVRQNAC+S and RQNAC significantly decreasedprocollagen Iα1 secretion in all three donors. Individually, N-acetylcysteine was shown to significantly decrease procollagen Iα1 secretionin two of the three donors. Valine significantly increased procollagenIα1 secretion in only one of two donors, while isoleucine and argininesignificantly increased procollagen Iα1 secretion in two of threedonors. In other words, glutamine administered individually did not havea significant impact on procollagen Iα1 secretion. As such, thereduction of the profibrogenic effect of LIV with arginine and glutaminerelative to that of LIV alone would not have been expected based on theeffect of individual amino acid treatments.

TABLE 70 Fold change of procollagen 1α1 secretion after administrationof an amino acid composition in a first donor Procollagen Iα1 Secretion(Fold Change of 1X) Std. Number Amino Acid Conc. Devia- of P- Signif-Supplement (X) Mean tion values value* icance LIVRQNAC 40 0.6283 0.05853 *** 0.0001 LIVRQNAC 30 0.5975 0.0709 3 **** 0.0001 LIVRQNAC 20 0.65040.0622 4 *** 0.0001 LIVRQNAC 10 0.8287 0.0936 4 * 0.0277 LIVRQNAC 11.0000 0.0908 4 LIVRQNAC + G 40 0.5288 0.0402 3 *** 0.0006 LIVRQNAC + G30 0.6297 0.0200 3 ** 0.0042 LIVRQNAC + G 20 0.5926 0.0634 4 ** 0.001 LIVRQNAC + G 10 0.7404 0.0920 4 * 0.0267 LIVRQNAC + G 1 1.0000 0.2151 4LIVRQNAC + S 40 0.5900 0.0450 3 *** 0.0003 LIVRQNAC + S 30 0.5562 0.12423 *** 0.0002 LIVRQNAC + S 20 0.6844 0.0638 3 ** 0.0022 LIVRQNAC + S 100.7003 0.0946 3 ** 0.0032 LIVRQNAC + S 1 1.0000 0.0311 3 LIV 40 1.30170.1474 3 ns 0.0518 LIV 30 1.3358 0.1922 3 * 0.0305 LIV 20 1.2592 0.07473 ns 0.0997 LIV 10 1.0149 0.1089 3 ns 0.9997 LIV 1 1.0000 0.0828 3 LIVRQ40 1.0070 0.1716 3 ns 0.9999 LIVRQ 30 1.0190 0.1103 3 ns 0.9983 LIVRQ 201.1403 0.0516 3 ns 0.3875 LIVRQ 10 1.0454 0.0908 3 ns 0.9609 LIVRQ 11.0000 0.0935 3 RQNAC 40 0.3622 0.0166 3 **** 0.0001 RQNAC 30 0.42320.0819 3 **** 0.0001 RQNAC 20 0.5819 0.0574 3 *** 0.0001 RQNAC 10 0.81810.0703 3 * 0.0313 RQNAC 1 1.0000 0.0967 3 N-Acetyl Cysteine 40 0.50760.0154 3 **** 0.0001 N-Acetyl Cysteine 20 0.6593 0.0914 3 *** 0.0003N-Acetyl Cysteine 10 0.7939 0.0715 3 ** 0.01  N-Acetyl Cysteine 5 0.91750.0519 3 ns 0.3855 N-Acetyl Cysteine 0 1.0000 0.0686 3

TABLE 71 Fold change of procollagen 1α1 secretion after administrationof a single amino acid composition in the first donor Procollagen Iα1Secretion (Fold Change of 1X) Std. Number Amino Acid Conc. Devia- of P-Signif- Supplement (μM) Mean tion values value* icance Valine 234201.2139 0.0544 3 ns 0.1392 Valine 11710 1.2069 0.0881 3 ns 0.155 Valine4684 1.1203 0.1908 3 ns 0.5111 Valine 234 1.0000 0.1389 4 Arginine 54401.0646 0.0939 3 ns 0.4155 Arginine 2720 1.1757 0.0466 3 * 0.01 Arginine1088 1.0291 0.0615 4 ns 0.8428 Arginine 109 1.0000 0.0389 4 Glutamine22484 1.0564 0.1293 3 ns 0.8468 Glutamine 11242 1.0888 0.0261 3 ns0.5648 Glutamine 3747 1.0757 0.1003 4 ns 0.6356 Glutamine 749 0.97900.0836 4 ns 0.993 Glutamine 562 1.0000 0.0596 3 Isoleucine 6639 1.21440.1129 3 ns 0.0537 Isoleucine 3320 1.1366 0.0938 3 ns 0.2411 Isoleucine1328 0.9229 0.0614 3 ns 0.6321 Isoleucine 66 1.0000 0.0953 3 Leucine15270 1.1710 0.1043 3 ns 0.094 Leucine 7635 1.0915 0.0832 3 ns 0.4736Leucine 3054 1.1410 0.1245 4 ns 0.1424 Leucine 153 1.0000 0.0481 4

TABLE 71-1 Fold change of procollagen 1α1 secretion after administrationof an amino acid composition in the second donor Procollagen Iα1Secretion (Fold Change of 1X) Std. Number Amino Acid Conc. Devia- of P-Signif- Supplement (X) Mean tion values value* icance LIVRQNAC 40 0.74650.0551 3 ** 0.0041 LIVRQNAC 30 0.6829 0.0991 3 *** 0.0007 LIVRQNAC 200.6922 0.0281 4 *** 0.0004 LIVRQNAC 10 0.7879 0.0748 4 ** 0.0085LIVRQNAC 1 1.0000 0.1141 4 LIVRQNAC + G 40 0.6372 0.0267 3 **** 0.0001LIVRQNAC + G 30 0.7347 0.0324 3 **** 0.0001 LIVRQNAC + G 20 0.67160.0552 4 **** 0.0001 LIVRQNAC + G 10 0.7823 0.0579 4 *** 0.0001LIVRQNAC + G 1 1.0000 0.0580 4 LIVRQNAC + S 40 0.8756 0.0372 3 ns 0.1229LIVRQNAC + S 30 0.7340 0.0432 3 ** 0.0019 LIVRQNAC + S 20 0.7405 0.04913 ** 0.0022 LIVRQNAC + S 10 0.7472 0.0710 3 ** 0.0027 LIVRQNAC + S 11.0000 0.1031 3 LIV 40 1.4409 0.0697 3 **** 0.0001 LIV 30 1.3679 0.01563 *** 0.0001 LIV 20 1.3418 0.1090 3 *** 0.0002 LIV 10 1.2176 0.0343 3 **0.0057 LIV 1 1.0000 0.0396 3 LIVRQ 40 0.9851 0.0534 3 ns 0.9965 LIVRQ 301.0185 0.0735 3 ns 0.9921 LIVRQ 20 0.9212 0.0215 3 ns 0.4893 LIVRQ 100.9558 0.0580 3 ns 0.8556 LIVRQ 1 1.0000 0.1134 3 RQNAC 40 0.6363 0.04323 *** 0.0002 RQNAC 30 0.6154 0.0196 3 *** 0.0001 RQNAC 20 0.7060 0.08513 *** 0.0009 RQNAC 10 0.8385 0.0248 3 * 0.041  RQNAC 1 1.0000 0.1071 3N-Acetyl Cysteine 40 0.8383 0.0378 3 ns 0.4053 N-Acetyl Cysteine 200.7378 0.1347 3 ns 0.1002 N-Acetyl Cysteine 10 0.8877 0.2282 3 ns 0.6842N-Acetyl Cysteine 5 0.8387 0.0832 3 ns 0.407  N-Acetyl Cysteine 0 1.00000.0808 3

TABLE 71-2 Fold change of procollagen 1α1 secretion after administrationof a single amino acid composition in the second donor Procollagen Iα1Secretion (Fold Change of 1X) Std. Number Amino Acid Conc. Devia- of P-Signif- Supplement (μM) Mean tion values value* icance Valine 234201.3068 0.0963 3 ** 0.0019 Valine 11710 1.2877 0.1122 3 ** 0.0029 Valine4684 1.2865 0.0717 4 ** 0.0018 Valine 234 1.0000 0.0589 4 Arginine 54401.1304 0.0187 3 ns 0.0937 Arginine 2720 1.0722 0.0791 3 ns 0.4483Arginine 1088 1.0126 0.0822 4 ns 0.989 Arginine 109 1.0000 0.0778 4Glutamine 22484 0.7143 0.0566 3 ** 0.0058 Glutamine 11242 0.7080 0.02463 ** 0.005  Glutamine 3747 0.7541 0.0860 4 * 0.0102 Glutamine 749 0.91910.1171 4 ns 0.5776 Glutamine 562 1.0000 0.1003 3 Isoleucine 6639 1.54230.1489 3 ** 0.006  Isoleucine 3320 1.4940 0.0238 3 * 0.0102 Isoleucine1328 1.4811 0.2307 3 * 0.0117 Isoleucine 66 1.0000 0.1264 3 Leucine15270 0.9518 0.0406 3 ns 0.9292 Leucine 7635 1.2628 0.1763 3 ns 0.0607Leucine 3054 1.0781 0.1735 4 ns 0.7374 Leucine 153 1.0000 0.0681 4

TABLE 71-3 Fold change of procollagen 1α1 secretion after administrationof an amino acid composition in the third donor Procollagen Iα1Secretion (Fold Change of 1X) Std. Number Amino Acid Conc. Devia- of P-Signif- Supplement (X) Mean tion values value* icance LIVRQNAC 40 0.90520.0344 3 ns 0.5685 LIVRQNAC 30 0.7456 0.0895 3 * 0.0192 LIVRQNAC 200.7817 0.0680 4 * 0.03  LIVRQNAC 10 0.9774 0.1451 4 ns 0.9927 LIVRQNAC 11.0000 0.1116 4 LIVRQNAC + G 40 0.7040 0.0080 3 ** 0.002  LIVRQNAC + G30 0.6249 0.0819 3 *** 0.0003 LIVRQNAC + G 20 0.6863 0.1334 4 *** 0.0006LIVRQNAC + G 10 1.0068 0.0642 4 ns 0.9998 LIVRQNAC + G 1 1.0000 0.0724 4LIVRQNAC + S 40 0.9190 0.0772 3 ns 0.3351 LIVRQNAC + S 30 0.8107 0.05963 * 0.0101 LIVRQNAC + S 20 0.8878 0.0129 3 ns 0.1296 LIVRQNAC + S 100.9814 0.0458 3 ns 0.9852 LIVRQNAC + S 1 1.0000 0.0780 3 LIV 40 1.32330.0667 3 ** 0.0024 LIV 30 1.2510 0.1070 3 * 0.0125 LIV 20 1.2702 0.06393 ** 0.0079 LIV 10 1.1912 0.1049 3 ns 0.0532 LIV 1 1.0000 0.0521 3 LIVRQ40 1.2020 0.1119 3 ns 0.1081 LIVRQ 30 1.1380 0.0955 3 ns 0.3407 LIVRQ 200.9489 0.1179 3 ns 0.9263 LIVRQ 10 1.0786 0.0764 3 ns 0.7564 LIVRQ 11.0000 0.1056 3 RQNAC 40 0.6590 0.0860 3 ** 0.0012 RQNAC 30 0.67080.0407 3 ** 0.0016 RQNAC 20 0.9135 0.1192 3 ns 0.5063 RQNAC 10 0.87830.0515 3 ns 0.245  RQNAC 1 1.0000 0.0740 3 N-Acetyl Cysteine 40 0.69620.0189 3 * 0.0125 N-Acetyl Cysteine 20 0.8521 0.0709 3 ns 0.2666N-Acetyl Cysteine 10 0.9391 0.1250 3 ns 0.8641 N-Acetyl Cysteine 51.0897 0.1245 3 ns 0.6511 N-Acetyl Cysteine 0 1.0000 0.1133 3

TABLE 71-4 Fold change of procollagen 1α1 secretion after administrationof a single amino acid composition in the third donor Procollagen Iα1Secretion (Fold Change of 1X) Std. Number Amino Acid Conc. Devia- of P-Signif- Supplement (μM) Mean tion values value* icance Valine 234201.1139 0.1077 3 ns 0.5315 Valine 11710 1.0498 0.1773 3 ns 0.918  Valine4684 1.0428 0.1036 4 ns 0.9323 Valine 234 1.0000 0.1203 4 Arginine 54401.2125 0.0862 3 * 0.0112 Arginine 2720 1.1314 0.0820 3 ns 0.1114Arginine 1088 1.0623 0.0629 4 ns 0.5378 Arginine 109 1.0000 0.0760 4Glutamine 22484 1.0121 0.0730 3 ns 0.9989 Glutamine 11242 1.1204 0.10563 ns 0.2356 Glutamine 3747 0.9734 0.0900 4 ns 0.9747 Glutamine 7491.0317 0.0644 4 ns 0.9538 Glutamine 562 1.0000 0.0447 3 Isoleucine 66391.4465 0.0958 3 ** 0.0014 Isoleucine 3320 1.2703 0.0352 3 * 0.024 Isoleucine 1328 1.2687 0.0374 3 * 0.0247 Isoleucine 66 1.0000 0.1629 3Leucine 15270 0.9892 0.0260 3 ns 0.9979 Leucine 7635 1.2027 0.0693 3 ns0.0638 Leucine 3054 1.1399 0.1385 4 ns 0.1844 Leucine 153 1.0000 0.10774

Example 13 Cytokine Secretion in Primary Human Macrophages

Isolation of Peripheral Blood Mononuclear Cell (PBMC)

Unpurified buffy coats (Research Blood Components) were carefully pouredinto 50 mL centrifuge tubes and diluted with room temperature Dulbecco'sPhosphate Buffered Saline (dPBS) with Calcium and Magnesium (Gibco).Diluted buffy coats were further divided into four total 50 mLcentrifuge tubes at 20 mL per tube. Lymphocyte Separation Medium(Corning) was carefully pipetted to the bottom of each centrifuge tube.Mixtures were centrifuged at 850×g for 32 minutes at 20° C. with 0deceleration and acceleration.

The PBMC layer was separated from other components after centrifugationand added to new 50 mL centrifuge tube containing 25 mL dPBS. Totalvolume was brought up to 50 mL with dPBS and centrifuged at 600×g for 10minutes at 20° C. with acceleration of 9, deceleration of 5. Supernatantwas carefully removed from cell pellets. The cell pellets wereresuspended using 10 mL dPBS. Total volume was then brought up to 50 mLusing dPBS and centrifuged at 450×g for 5 min at 20° C. withacceleration of 9, deceleration of 9. The supernatant removal and cellpellet resuspension was repeated again.

The supernatant was then carefully removed from cell pellets. Cellpellets were resuspended in 10 mL dPBS without calcium or magnesium andfiltered through a 70 uM cell strainer. The total PBMC number wasdetermined using a Cellometer K2 automated cell counter. A total of 5E6cells were saved for flow cytometric analysis. Remaining cells werecentrifuged at 490×g for 5 minutes at 20° C. with acceleration of 9,deceleration of 9.

CD14+ Cell Selection

CD14+ cells were selected using EasySep™ Human CD14 Positive SelectionKit II (STEMCELL Technologies). Cells were resuspended in cold EasySep™Buffer (STEMCELL Technologies) at 1×10⁸ cells/mL. A total of 100 uL/mLEasySep™ Human CD14 Positive Selection Cocktail II was added to the cellsuspension, mixed, and incubated at room temperature for 10 minutes. Atotal of 100 uL/mL RapidSpheres were added to the mixture and incubatedat room temperature for 3 minutes after mixing, then RoboSep buffer wasadded to bring up the total volume to 10 mL. The mixture in a 15 mL tubewas placed in magnet and incubated at room temperature for 3 minutes.Supernatant was discarded and 10 mL fresh EasySep™ buffer was added to15 mL tube. The addition of RoboSep buffer, mixing, and discarding ofsupernatant was was repeated two more times.

Negative and positive fractions were centrifuged at 490×g for 5 minutesat 20° C. with acceleration of 9, deceleration of 9, and resuspended inDMEM (Gibco) and 10% Heat Inactivated Fetal Bovine Serum (Atlanta Bio)and Penicillin/Streptomycin. Cells were counted and centrifuged again at490×g for 5 minutes at 20° C. with acceleration of 9, deceleration of 9.After centrifugation, cell were resuspended in DMEM (Gibco) and 10% HeatInactivated Fetal Bovine Serum (Atlanta Bio) and Penicillin/Streptomycincontaining 500 U/mL GM- and plated at 1-2×10⁶ cells/mL on 10 cm tissueculture plates. Cells were kept in 37° C., 5% CO₂ in betweenfeedings/harvest.

CD14+ Cell Feeding

Cells were fed every 3-4 days by removing media and unattached cells,centrifuging at 490×g for 5 minutes at 20 C with acceleration of 9,deceleration of 9, and resuspending in fresh DMEM (Gibco) and 10% HeatInactivated Fetal Bovine Serum (Atlanta Bio) andPenicillin/Streptomycincontaining 500 U/mL GM-CSF. Resuspended cellswere seeded back onto 10 cm tissue culture plates and incubated at 37°C., 5% CO₂.

Macrophage Harvest

After complete cell attachment, culture supernatant was removed andcultures were washed 1× with 5 mL PBS. A total of 3 mL room temperatureCellstripper was added and cultures were incubated at 37° C., 5% CO₂ forapproximately 10 minutes until cells were rounded and beginning todetach. Cell scraper was used to completely detach cells from plate.Collected cell were spun down at 490 g for 5 min at room temperature andresuspended in 10% DMSO in Heat Inactivated Fetal Bovine Serum andimmediately frozen in −80 C.

Screen

Primary human PMBC derived macrophages were seeded on day 0 at 3.0E4cells per well in 96-well microplates (ThermoFisher) in Dulbecco'sModified Eagle Medium (DMEM) (Gibco) supplemented withpenicillin-streptomycin (Hyclone) and 10% heat inactivated fetal bovineserum (HI-FBS) (Atlanta Bio) and incubated overnight at 37° C., 5% CO2.On day 1, cells were washed once with 150 uL per well DPBS (Gibco) andtreated with 75 uL of:

-   -   a. Amino acid free DMEM (US Biologicals) containing a defined        custom amino acid concentration based on the mean physiological        concentrations in blood based on values published in the Human        Metabolome Database (HMDB), with 6 mM glucose, 1 mM sodium        pyruvate, 10 mM HEPES, 0.2% primocin (InVivoGen); or    -   b. The same medium described above with one amino acid at        various concentrations including complete dropout.

On day 2, cells were treated with 75 uL of the same mediums describedabove supplemented with 0.30 ng/mL lipopolysaccharide (LPS) (Sigma) fora final concentration of 0.15 ng/mL LPS. Control wells were treated with1 uM BX-795 (Tocis), 1 uM TAK242 (Sigma), 0.15 ng/mL LPS, or phosphatebuffered saline (PBS).

On day 3, the supernatant was collected and immediately frozen in −80°C. freezer. Cells were washed once with 150 uL DPBS and viability wasassessed using the WST-8 Cell Proliferation Cytotoxicity Assay(Dojindo). Following the assay, cells were washed twice with 150 uL PBSand fixed with 4% paraformaldehyde for 5 min followed by two additionalwashes with 150 uL PBS. Protein levels in supernatant samples wereanalyzed by ELISA for IL-6 and TNFa using commercially available kits(R&D Systems) according to manufacturer-supplied protocols. Results areshown in Tables 71-5 through 71-10 below.

TABLE 71-5 IL-6 Measurements: Donor 1 Donor 1 IL-6 Measurements Std.Number Amino Acid Conc. Devia- of P- Signif- Supplement (X) Mean tionvalues value* icance LIVRQNAC 40 −619.787 114.1592 3 0.0001 ****LIVRQNAC 30 −525.849 63.87122 3 0.0001 **** LIVRQNAC 20 −290.199 33.55843 0.0092 ** LIVRQNAC 10 51.81434 183.3933 3 0.9479 ns LIVRQNAC 1 0148.7761 3 na na LIVRQNAC + G 40 −1099.11 44.1139 3 0.0001 ****LIVRQNAC + G 30 −903.836 107.7113 3 0.0001 **** LIVRQNAC + G 20 −616.626114.7826 3 0.0001 **** LIVRQNAC + G 10 −367.918 98.44611 3 0.0007 ***LIVRQNAC + G 1 0 172.9553 3 na na LIVRQNAC + S 40 −968.997 90.53282 30.0001 **** LIVRQNAC + S 30 −798.326 52.89122 3 0.0001 **** LIVRQNAC + S20 −506.804 63.85224 3 0.0001 **** LIVRQNAC + S 10 −243.259 114.742 30.0365 * LIVRQNAC + S 1 0 259.8506 3 na na LIV 40 4.918642 62.7077 30.9999 ns LIV 30 86.01907 128.1151 3 0.7604 ns LIV 20 112.1501 83.624363 0.564 ns LIV 10 54.22668 63.10515 3 0.9392 ns LIV 1 0 75.98804 3 na naLIVRQ 40 322.0706 73.87715 3 0.0033 ** LIVRQ 30 297.8004 34.60168 30.0072 ** LIVRQ 20 604.021 203.8836 3 0.0001 **** LIVRQ 10 289.179857.78952 3 0.0095 ** LIVRQ 1 0 93.58494 3 na na RQNAC 40 −911.01112.65475 3 0.0001 **** RQNAC 30 −766.912 26.23659 3 0.0001 **** RQNAC 20−511.403 32.15983 3 0.0001 **** RQNAC 10 −201.63 6.477522 3 0.1054 nsRQNAC 1 0 174.9658 3 na na N-Acetyl Cysteine 40 −914.194 56.77271 30.0001 **** N-Acetyl Cysteine 20 −553.802 85.27013 3 0.0001 ****N-Acetyl Cysteine 10 −121.142 53.05191 3 0.4973 ns N-Acetyl Cysteine 5308.1772 263.4651 3 0.0052 ** N-Acetyl Cysteine 0 0 45.08485 3 na naDonor 1 IL-6 Measurements Std. Number Amino Acid Conc. Devia- of P-Signif- Supplement (μM) Mean tion values value* icance Valine 23420−106.268 155.3559 3 0.7885 ns Valine 11710 −97.25 77.26313 3 0.8339 nsValine 4684 −85.9843 74.99317 3 0.8841 ns Valine 234 0 124.8497 3 na naArginine 5440 357.4394 154.8508 3 0.0159 * Arginine 2720 −186.5785.86105 3 0.3477 ns Arginine 1088 −181.36 131.6475 3 0.3722 ns Arginine109 0 282.0306 3 na na Glutamine 22484 440.1437 114.443 3 0.0022 **Glutamine 11242 397.1745 23.36272 3 0.0064 ** Glutamine 3747 291.544381.30853 3 0.0623 ns Glutamine 749 0 73.06692 3 na na Isoleucine 6639−218.332 146.5098 3 0.221 ns Isoleucine 3320 −15.8843 89.88616 3 0.9998ns Isoleucine 1328 25.98372 323.6109 3 0.9984 ns Isoleucine 66 048.21125 3 na na Leucine 15270 84.46122 68.15253 3 0.8902 ns Leucine7635 −69.9873 99.00843 3 0.9398 ns Leucine 3054 244.9743 355.6551 30.1442 ns Leucine 153 0 61.85589 3 na naTreatment with LIVRQNAC, LIVRQNAC+G, LIVRQNAC+S, RQNAC, and NACsignificantly reduced LPS-induced IL-6 secretion in primary humanmonocyte-derived macrophages. Treatment with LIVRQ significantlyincreased IL-6 secretion, while LIV had no effect. Arginine andglutamine administered alone increased IL-6 secretion while other aminoacids alone did not effect IL-6 secretion. Two Way ANOVA DunnettMultiple Comparisons was performed for statistical analysis. Mean valuesrepresented as baseline subtracted values.

TABLE 71-6 IL-6 Measurements: Donor 2 Donor 2 IL-6 Measurements Std.Number Amino Acid Conc. Devia- of P- Signif- Supplement (X) Mean tionvalues value* icance LIVRQNAC 40 −27.1916 1.853569 3 0.0003 *** LIVRQNAC30 −21.5766 1.709414 3 0.0045 ** LIVRQNAC 20 −8.20655 8.458638 3 0.5143ns LIVRQNAC 10 −1.71581 6.104437 3 0.9965 ns LIVRQNAC 1  −2.4E−1511.85079 3 LIVRQNAC + G 40 −33.2001 3.55425 3 0.0001 **** LIVRQNAC + G30 −30.8468 0.854995 3 0.0001 **** LIVRQNAC + G 20 −18.4318 4.870421 30.0187 * LIVRQNAC + G 10 14.63551 21.82024 3 0.0824 ns LIVRQNAC + G 1  2.37E−15 8.607557 3 LIVRQNAC + S 40 −26.5993 2.963677 3 0.0004 ***LIVRQNAC + S 30 −14.2166 1.460268 3 0.0954 ns LIVRQNAC + S 20 −8.25222.917345 3 0.5095 ns LIVRQNAC + S 10 8.127841 1.783214 3 0.5227 nsLIVRQNAC + S 1 0 6.232673 3 LIV 40 34.10306 1.950493 3 0.0001 **** LIV30 31.10835 9.757211 3 0.0001 **** LIV 20 20.32684 3.17293 3 0.0081 **LIV 10 15.10204 9.179111 3 0.0697 ns LIV 1  −7.1E−15 4.738966 3 LIVRQ 4049.62156 17.37012 3 0.0001 **** LIVRQ 30 42.9625 7.798872 3 0.0001 ****LIVRQ 20 48.38603 13.08566 3 0.0001 **** LIVRQ 10 45.99191 15.19687 30.0001 **** LIVRQ 1   1.18E−15 6.324379 3 RQNAC 40 −36.5521 1.877658 30.0001 **** RQNAC 30 −26.3768 0.744676 3 0.0004 *** RQNAC 20 −18.74281.353649 3 0.0164 * RQNAC 10 −3.74427 4.74578 3 0.9393 ns RQNAC 1  2.37E−15 12.26314 3 N-Acetyl Cysteine 40 −33.7585 0.895842 3 0.0001**** N-Acetyl Cysteine 20 −24.9999 1.083467 3 0.0008 *** N-AcetylCysteine 10 −9.75111 2.381012 3 0.3617 ns N-Acetyl Cysteine 5 −0.794585.988677 3 0.9998 ns N-Acetyl Cysteine 0  −2.4E−15 1.900091 3 Donor 2IL-6 Measurements Std. Number Amino Acid Conc. Devia- of P- Signif-Supplement (μM) Mean tion values value* icance Valine 23420 4.39589910.35903 3 0.973 ns Valine 11710 −1.19605 7.303571 3 0.9998 ns Valine4684 −4.52846 4.069907 3 0.97 ns Valine 234  −4.7E−15 9.361734 3Arginine 5440 −12.4164 0.292618 3 0.5017 ns Arginine 2720 −13.61022.1177 3 0.4207 ns Arginine 1088 −9.70116 9.286942 3 0.6995 ns Arginine109   2.37E−15 14.30728 3 Glutamine 22484 34.38845 7.467725 3 0.0026 **Glutamine 11242 63.31441 35.02748 3 0.0001 **** Glutamine 3747 22.515439.686139 3 0.0721 ns Glutamine 749   2.37E−15 2.203881 3 Isoleucine 6639−1.77438 10.22772 3 0.999 ns Isoleucine 3320 2.305485 1.328015 3 0.9975ns Isoleucine 1328 −2.31776 9.121049 3 0.9974 ns Isoleucine 66 0 12.34133 Leucine 15270 47.59735 16.64049 3 0.0001 **** Leucine 7635 30.460657.144005 3 0.0087 ** Leucine 3054 29.60609 13.39676 3 0.0111 * Leucine153   7.11E−15 6.308577 3Treatment with LIVRQNAC, LIVRQNAC+G, LIVRQNAC+S, RQNAC, and NACsignificantly reduced LPS-induced IL-6 secretion in primary humanmonocyte-derived macrophages. Treatment with LIVRQ and LIV significantlyincreased IL-6 secretion. Glutamine and leucine administered aloneincreased IL-6 secretion, while the other amino acids alone had noeffect. Two Way ANOVA Dunnett Multiple Comparisons was performed forstatistical analysis. Mean values represented as baseline subtractedvalues.

TABLE 71-7 IL-6 Measurements: Donor 3 Donor 3 IL-6 Measurements Std.Number Amino Acid Conc. Devia- of P- Signif- Supplement (X) Mean tionvalues value* icance LIVRQNAC 40 −18.2445 4.129349 3 0.7529 ns LIVRQNAC30 −16.8219 1.366045 3 0.8001 ns LIVRQNAC 20 −13.4826 12.48206 3 0.8948ns LIVRQNAC 10 −34.4539 37.38053 3 0.2356 ns LIVRQNAC 1  −1.4E−1414.03982 3 LIVRQNAC + G 40 −54.4799 5.467815 3 0.0228 * LIVRQNAC + G 30−48.3118 1.960574 3 0.0513 ns LIVRQNAC + G 20 −55.792 7.763897 3 0.019 *LIVRQNAC + G 10 −44.8309 14.34972 3 0.0783 ns LIVRQNAC + G 1 0 26.014713 LIVRQNAC + S 40 −14.5337 15.82418 3 0.868 ns LIVRQNAC + S 30 −25.912710.00119 3 0.479 ns LIVRQNAC + S 20 −25.8862 21.61536 3 0.48 nsLIVRQNAC + S 10 −11.9742 10.3333 3 0.9277 ns LIVRQNAC + S 1  −4.3E−1415.34164 3 LIV 40 10.21257 37.58938 3 0.9576 ns LIV 30 −32.6891 24.862 30.2771 ns LIV 20 27.66715 39.40901 3 0.4207 ns LIV 10 9.44789 71.20002 30.9677 ns LIV 1  −4.7E−14 27.50075 3 LIVRQ 40 74.9145 12.55033 3 0.001*** LIVRQ 30 120.1764 20.21514 3 0.0001 **** LIVRQ 20 77.12007 11.454523 0.0007 *** LIVRQ 10 67.95483 43.58345 3 0.003 ** LIVRQ 1  −2.4E−1427.62048 3 RQNAC 40 −45.9765 5.740028 3 0.0683 ns RQNAC 30 −53.384516.45009 3 0.0265 * RQNAC 20 −65.6761 3.400465 3 0.0044 ** RQNAC 10−32.8776 33.99103 3 0.2724 ns RQNAC 1  −2.8E−14 23.14404 3 N-AcetylCysteine 40 −140.851 4.662272 3 0.0001 **** N-Acetyl Cysteine 20−122.656 8.219985 3 0.0001 **** N-Acetyl Cysteine 10 −103.586 28.4385 30.0001 **** N-Acetyl Cysteine 5 −70.3269 8.563896 3 0.0021 ** N-AcetylCysteine 0  −9.5E−15 11.75797 3 Donor 3 IL-6 Measurements Std. NumberAmino Acid Conc. Devia- of P- Signif- Supplement (μM) Mean tion valuesvalue* icance Valine 23420 −29.2004 25.98066 3 0.4329 ns Valine 11710−43.8022 8.331697 3 0.1239 ns Valine 4684 −30.0609 8.478329 3 0.4072 nsValine 234   4.26E−14 17.2027 3 Arginine 5440 −6.80983 0.643932 3 0.9922ns Arginine 2720 −7.50318 22.06663 3 0.9888 ns Arginine 1088 31.578670.48311 3 0.3642 ns Arginine 109 0 17.26952 3 Glutamine 22484 108.515855.59202 3 0.0001 **** Glutamine 11242 98.4903 58.37 3 0.0001 ****Glutamine 3747 25.35457 16.40416 3 0.556 ns Glutamine 749   3.79E−1416.54987 3 Isoleucine 6639 −16.3663 8.09174 3 0.9718 ns Isoleucine 33200 19.80362 3 0.9928 ns Isoleucine 1328 −28.9897 13.10903 3 0.6593 nsIsoleucine 66 −6.69039 13.72995 3 Leucine 15270 # # 3 # # Leucine 7635 ## 3 # # Leucine 3054 # # 3 # # Leucine 153 # # 3 # # # Leucine was notmeasured in Exp3 due to technical errorTreatment with LIVRQNAC+G, LIVRQNAC+S, RQNAC, and NAC significantlyreduced LPS-induced IL-6 secretion in primary human monocyte-derivedmacrophages. Treatment with LIVRQ increased IL-6 secretion, while LIVand LIVRQNAC had no statistically significant effects on IL-6 secretion.Glutamine administered alone significantly increased IL-6 secretion,while other amino acids alone had no effect. Two Way ANOVA DunnettMultiple Comparisons was performed for statistical analysis. Mean valuesrepresented as baseline subtracted values.

TABLE 71-8 TNFalpha Measurements: Donor 1 Donor 1 TNFa Measurements Std.Number Amino Acid Conc. Devia- of P- Signif- Supplement (X) Mean tionvalues value* icance LIVRQNAC 40 −422.74 4.347575 3 0.0001 **** LIVRQNAC30 −389.74 1.004633 3 0.0001 **** LIVRQNAC 20 −336.69 3.007435 3 0.0001**** LIVRQNAC 10 −246.04 27.61929 3 0.0001 **** LIVRQNAC 1 0 36.31082 3LIVRQNAC + G 40 −490.92 4.427614 3 0.0001 **** LIVRQNAC + G 30 −447.739.819865 3 0.0001 **** LIVRQNAC + G 20 −377.32 5.837159 3 0.0001 ****LIVRQNAC + G 10 −288.29 9.642365 3 0.0001 **** LIVRQNAC + G 1 0 37.443533 LIVRQNAC + S 40 −415.03 4.800449 3 0.0001 **** LIVRQNAC + S 30 −379.444.694868 3 0.0001 **** LIVRQNAC + S 20 −323.77 7.971135 3 0.0001 ****LIVRQNAC + S 10 −209.59 21.15676 3 0.0001 **** LIVRQNAC + S 1 0 30.04923 LIV 40 60.37 20.26331 3 0.0065 ** LIV 30 42.09 22.95664 3 0.0865 nsLIV 20 63.37 37.24144 3 0.004 ** LIV 10 45.61 44.71078 3 0.0556 ns LIV 10 10.49958 3 LIVRQ 40 6.38 17.1283 3 0.9909 ns LIVRQ 30 −6.72 18.9622 30.989 ns LIVRQ 20 38.38 39.85515 3 0.1333 ns LIVRQ 10 −18.95 10.84371 30.6982 ns LIVRQ 1 0 36.96184 3 RQNAC 40 −408.44 1.179877 3 0.0001 ****RQNAC 30 −390.41 1.341282 3 0.0001 **** RQNAC 20 −338.2 3.284307 30.0001 **** RQNAC 10 −251.35 4.121085 3 0.0001 **** RQNAC 1 0 51.06933 3N-Acetyl Cysteine 40 −644.49 2.42197 3 0.0001 **** N-Acetyl Cysteine 20−561.33 8.435064 3 0.0001 **** N-Acetyl Cysteine 10 −446.88 12.22132 30.0001 **** N-Acetyl Cysteine 5 −326.24 11.10173 3 0.0001 **** N-AcetylCysteine 0 0 42.00516 3 Donor 1 TNFa Measurements Std. Number Amino AcidConc. Devia- of P- Signif- Supplement (μM) Mean tion values value*icance Valine 23420 −14.98 20.86784 3 0.9928 ns Valine 11710 −41.7736.61662 3 0.7784 ns Valine 4684 −40.37 32.31016 3 0.7974 ns Valine 2340 24.8661 3 Arginine 5440 62.06 48.80326 3 0.4786 ns Arginine 2720 5.1215.47951 3 0.9998 ns Arginine 1088 −24.33 17.74317 3 0.9577 ns Arginine109 0 18.5366 3 Glutamine 22484 −103.07 27.02483 3 0.0985 ns Glutamine11242 −65.24 23.02631 3 0.4346 ns Glutamine 3747 −45.7 28.56445 3 0.7222ns Glutamine 749 0 30.75138 3 Isoleucine 6639 −40.95 78.56369 3 0.7896ns Isoleucine 3320 −96.3 45.66981 3 0.1339 ns Isoleucine 1328 −42.6821.07739 3 0.7657 ns Isoleucine 66 0 115.9559 3 Leucine 15270 −46.2129.00402 3 0.7148 ns Leucine 7635 −23.04 40.08864 3 0.965 ns Leucine3054 42.04 77.19161 3 0.7746 ns Leucine 153 0 157.6578 3Treatment with LIVRQNAC, LIVRQNAC+G, LIVRQNAC+S, RQNAC, and NACsignificantly reduced LPS-induced TNFa secretion in primary humanmonocyte-derived macrophages. Treatment with LIV increased TNFasecretion, while LIVRQ had no significant effects on TNFa secretion.None of the individually administered amino acids had an effect on TNFasecretion. Two Way ANOVA Dunnett Multiple Comparisons was performed forstatistical analysis. Mean values represented as baseline subtractedvalues.

TABLE 71-9 TNFalpha Measurements: Donor 2 Donor 2 TNFa Measurements Std.Number Amino Acid Conc. Devia- of P- Signif- Supplement (X) Mean tionvalues value* icance LIVRQNAC 40 −98.1341 2.118962 3 0.0001 ****LIVRQNAC 30 −85.1019 1.385677 3 0.0001 **** LIVRQNAC 20 −64.336410.07525 3 0.0001 **** LIVRQNAC 10 −38.3512 5.120689 3 0.0001 ****LIVRQNAC 1 0 5.45587 3 LIVRQNAC + G 40 −91.3454 5.994009 3 0.0001 ****LIVRQNAC + G 30 −82.4397 4.200763 3 0.0001 **** LIVRQNAC + G 20 −61.2478.702492 3 0.0001 **** LIVRQNAC + G 10 −23.9913 7.471422 3 0.008 **LIVRQNAC + G 1  −4.7E−15 4.578295 3 LIVRQNAC + S 40 −74.1572 4.163823 30.0001 **** LIVRQNAC + S 30 −64.0016 5.549308 3 0.0001 **** LIVRQNAC + S20 −47.5673 3.970363 3 0.0001 **** LIVRQNAC + S 10 −28.635 7.390447 30.0012 ** LIVRQNAC + S 1  −4.7E−15 7.564883 3 LIV 40 49.84155 4.092799 3**** 0.0001 LIV 30 29.1118 14.72509 3 *** 0.001 LIV 20 30.17595 5.7975183 *** 0.0006 LIV 10 16.68974 10.85983 3 ns 0.0974 LIV 1 0 10.41523 3LIVRQ 40 64.1705 27.82953 3 **** 0.0001 LIVRQ 30 50.92104 6.955429 3**** 0.0001 LIVRQ 20 45.65882 19.0128 3 **** 0.0001 LIVRQ 10 32.3703819.44425 3 *** 0.0002 LIVRQ 1  −4.7E−15 5.942707 3 RQNAC 40 −84.1475.821583 3 **** 0.0001 RQNAC 30 −77.9626 1.626776 3 **** 0.0001 RQNAC 20−63.3754 3.494595 3 **** 0.0001 RQNAC 10 −37.6072 1.88043 3 **** 0.0001RQNAC 1  −9.5E−15 4.727924 3 N-Acetyl Cysteine 40 −103.984 0.720962 30.0001 **** N-Acetyl Cysteine 20 −88.6528 0.668195 3 0.0001 ****N-Acetyl Cysteine 10 −70.8382 12.08717 3 0.0001 **** N-Acetyl Cysteine 5−54.1596 11.06287 3 0.0001 **** N-Acetyl Cysteine 0   9.47E−15 2.9268813 Donor 2 TNFa Measurements Std. Number Amino Acid Conc. Devia- of P-Signif- Supplement (μM) Mean tion values value* icance Valine 23420−1.25079 12.85688 3 0.9991 ns Valine 11710 −0.83505 8.524018 3 0.9998 nsValine 4684 −0.00221 5.127759 3 0.9999 ns Valine 234  −4.7E−15 8.7173753 Arginine 5440 −0.57378 8.672536 3 0.9999 ns Arginine 2720 −3.763342.467885 3 0.9594 ns Arginine 1088 −12.7222 4.764842 3 0.2488 nsArginine 109   1.42E−14 3.511446 3 Glutamine 22484 11.50181 6.216029 30.3311 ns Glutamine 11242 20.03996 11.90208 3 0.0279 * Glutamine 37479.338214 9.748253 3 0.5134 ns Glutamine 749  −9.5E−15 7.275868 3Isoleucine 6639 19.25756 5.097831 3 0.0365 * Isoleucine 3320 10.260617.861148 3 0.4307 ns Isoleucine 1328 2.918887 1.921961 3 0.9836 nsIsoleucine 66   4.74E−15 6.264135 3 Leucine 15270 46.68507 11.63209 30.0001 **** Leucine 7635 41.97528 6.512087 3 0.0001 **** Leucine 305431.74019 11.56537 3 0.0002 *** Leucine 153 0 0.482598 3Treatment with LIVRQNAC, LIVRQNAC+G, LIVRQNAC+S, RQNAC, and NACsignificantly reduced LPS-induced TNFa secretion in primary humanmonocyte-derived macrophages. Treatment with LIV and LIVRQ increasedTNFa secretion. Leucine, isoleucine, and glutamine administeredindividually increased TNFa secretion, while the other amino acids hadno effect. Two Way ANOVA Dunnett Multiple Comparisons was performed forstatistical analysis. Mean values represented as baseline subtractedvalues.

TABLE 71-10 TNFalpha Measurements: Donor 3 Donor 3 TNFa MeasurementsStd. Number Amino Acid Conc. Devia- of P- Signif- Supplement (X) Meantion values value* icance LIVRQNAC 40 −18.7507 2.487301 3 0.0001 ****LIVRQNAC 30 −15.5979 0.932399 3 0.0006 *** LIVRQNAC 20 −10.7042 3.0135273 0.026 * LIVRQNAC 10 −8.49034 2.434812 3 0.1029 ns LIVRQNAC 1 04.067982 3 LIVRQNAC + G 40 −14.6552 3.149813 3 0.0013 ** LIVRQNAC + G 30−11.6973 2.026588 3 0.0129 * LIVRQNAC + G 20 −8.0218 0.671662 3 0.1331ns LIVRQNAC + G 10 −4.8035 1.658348 3 0.5453 ns LIVRQNAC + G 1  −2.4E−155.625453 3 LIVRQNAC + S 40 −14.247 1.800575 3 0.0018 ** LIVRQNAC + S 30−15.1388 1.568817 3 0.0009 *** LIVRQNAC + S 20 −12.4722 3.334857 30.0073 ** LIVRQNAC + S 10 −6.72057 1.833554 3 0.2549 ns LIVRQNAC + S 1 04.171555 3 LIV 40 14.07984 11.14252 3 0.002 ** LIV 30 1.759786 1.1027063 0.9748 ns LIV 20 14.51396 10.41503 3 0.0014 ** LIV 10 8.56095712.86074 3 0.0989 ns LIV 1   2.37E−15 3.660423 3 LIVRQ 40 25.844530.659584 3 0.0001 **** LIVRQ 30 33.74883 5.974096 3 0.0001 **** LIVRQ 2020.94481 2.163828 3 0.0001 **** LIVRQ 10 15.45187 3.942596 3 0.0007 ***LIVRQ 1 0 4.575346 3 RQNAC 40 −21.5102 1.191926 3 0.0001 **** RQNAC 30−20.8898 2.622446 3 0.0001 **** RQNAC 20 −19.9558 3.302225 3 0.0001 ****RQNAC 10 −9.09425 5.483242 3 0.0725 ns RQNAC 1 0 6.189505 3 N-AcetylCysteine 40 −55.3093 0.809363 3 0.0001 **** N-Acetyl Cysteine 20−48.4373 1.563179 3 0.0001 **** N-Acetyl Cysteine 10 −41.7266 3.533914 30.0001 **** N-Acetyl Cysteine 5 −33.6246 0.253484 3 0.0001 **** N-AcetylCysteine 0   4.74E−15 8.55997 3 Donor 3 TNFa Measurements Std. NumberAmino Acid Conc. Devia- of P- Signif- Supplement (μM) Mean tion valuesvalue* icance Valine 23420 3.688279 7.532913 3 0.8962 ns Valine 11710−2.59866 2.586099 3 0.9674 ns Valine 4684 0.126 0.903014 3 0.9999 nsValine 234  −2.4E−15 2.731283 3 Arginine 5440 −1.76662 4.067694 3 0.992ns Arginine 2720 −0.96691 4.86075 3 0.9991 ns Arginine 1088 3.13115310.346 3 0.9384 ns Arginine 109   3.55E−15 4.325877 3 Glutamine 2248429.14034 17.71417 3 0.0001 **** Glutamine 11242 18.00238 14.58602 30.0061 ** Glutamine 3747 1.935546 2.127977 3 0.9887 ns Glutamine 749 05.196592 3 Isoleucine 6639 −1.66019 4.262718 3 0.9938 ns Isoleucine 33203.308901 3.745411 3 0.9262 ns Isoleucine 1328 −6.22991 0.48195 3 0.5976ns Isoleucine 66  −2.4E−15 3.844593 3 Leucine 15270 # # 3 # # Leucine7635 # # 3 # # Leucine 3054 # # 3 # # Leucine 153 # # 3 # # # Leucinewas not measured in Exp3 due to technical errorTreatment with LIVRQNAC, LIVRQNAC+G, LIVRQNAC+S, RQNAC, and NACsignificantly reduced LPS-induced TNFa secretion in primary humanmonocyte-derived macrophages. Treatment with LIV and LIVRQ increasedTNFa secretion. Individually administered amino acids had no significanteffect on TNFa secretion, except for glutamine which increased TNFasecretion. Two Way ANOVA Dunnett Multiple Comparisons was performed forstatistical analysis. Mean values represented as baseline subtractedvalues.

Example 14 Treatment of NAFLD Patients with an Amino Acid Composition

The study described herein features the administration of a compositionincluding amino acids to treat patients with NAFLD. The composition caninclude about 2 g of leucine, about 1 g of isoleucine, about 1 g ofvaline, about 3.62 g of arginine, about 4 g of glutamine, and about 0.3g of N-acetylcysteine for administration three times per day (e.g., atotal of about 36 g per day). The composition can also include about 2 gof leucine, about 1 g of isoleucine, about 1 g of valine, about 3.62 gof arginine, about 4 g of glutamine, and about 0.6 g of N-acetylcysteinefor administration three times per day (e.g., a total of about 37 g perday).

Alternatively, the composition can include about 4 g of leucine, about 2g of isoleucine, about 2 g of valine, about 7.24 g of arginine, about 8g of glutamine, and about 0.6 g of N-acetylcysteine for administrationtwo or three times per day (e.g., a total of about 48 g or a total ofabout 72 g per day). The composition can also include about 4 g ofleucine, about 2 g of isoleucine, about 2 g of valine, about 7.24 g ofarginine, about 8 g of glutamine, and about 1.2 g of N-acetylcysteinefor administration two or three times per day (e.g., a total of about 49g or a total of about 73 g per day).

For each composition, the dose can be administered prior to,concurrently with, or following a meal. Alternatively, the compositionis not administered immediately before, with, or after a meal. The aminoacid composition can be administered for a period of at least 12 weeks,e.g., for 12 weeks, 13 weeks, 14 weeks, 15 weeks, or 16 weeks. Inparticular, the amino acid composition is administered for a period ofat least 16 weeks, e.g., for 16 weeks. The composition can beadministered orally.

Key criteria for selecting NAFLD patients for enrollment in a clinicalstudy of the amino acid composition include: 1) a diagnosis of NAFLD; 2)type 2 diabetes; 3) a relatively high BMI; 4) a NAFLD Fibrosis Score ofless than 0.6; 5) a liver biopsy; and 7) a MRI and/or CT assessment ofthe liver. The patients can have type 2 diabetes in addition to NAFLD.

Fatty liver disease can be document by a prior history of steatosisconfirmed within 3 months of screening by at least one of the followingmethods: liver fat by MRI with a PDFF≥8%; fibroscan with ControlAttenuation Parameter ≥300 dB/m; or liver biopsy indicating non-NASHNAFLD steatosis >Grade I. If the patient does not have this documentedprior history of steatosis within 3 months of screening, then a liverfat score of ≥10% must be documented at the time of screening using thefollowing formula:Predicted percent liver fat=10{circumflex over( )}(−0.805+(0.282*metabolic syndrome[yes=1/no=0])+(0.078*type 2diabetes[yes=2/no=0])+(0.525*log 10(insulin mU/L))+(0.521*log 10(ASTU/L))−(0.454*log 10(AST/ALT))³⁴

Patients can be on stable exercise, diet and lifestyle routine within 3months prior to screening, with no major body weight fluctuations, e.g.,subjects should be within ±3% of their body weight over the last 3months at the time of screening. Patients can have a body mass index(BMI)≥32 kg/m2 at screening. For sites whose MRI equipment cannotaccommodate a patient with a BMI of ≥45 kg/m2, an upper limit between 40to 45 kg/m2 may be applied.

Patients must be on a stable dose of glucose-lowering medication (whichcan include metformin, sulfonylureas, dipeptidyl peptidase-4 (DPP-4)inhibitors, sodium-glucose co-transporter 2 (SGLT2) inhibitors, orlong-acting basal insulin) for at least 3 months before Screening andplan to remain on the same medication without anticipated doseadjustments of their medications for the duration of the study. Patientsmay be included in the study if they are concurrently treated withanti-hypertensive medications (e.g., beta blockers, hydrochlorothiazide,ACE inhibitors, angiotensin receptor blockers), medications fordyslipidemia (e.g., statins, fibrates), and medication forhypothyroidism (e.g., levothyroxine), so long as they have been onstable doses and regimen of these medications for at least 3 monthsbefore Screening and plan to remain on the same medication withoutanticipated dose adjustments of their medications for the duration ofthe study. Patients may be on vitamin supplements (e.g. multivitamins;vitamin E<400 IU/day). However, they must be on stable doses and regimenof these vitamin supplements for at least 3 months before screeningwithout anticipated dose adjustments for the duration of the study.

Primary endpoints in the study include the safety and tolerability ofadministering the composition including amino acids to patients withNAFLD. Secondary endpoints indicative of an improvement in symptoms ofNAFLD include the following: 1) intrahepatic fat reduction by MRI; and2) assessment of biomarkers involved in liver biochemistry,fibrosis/apoptosis, and meteabolism. The following biomarkers can beassessed in a sample (e.g., a plasma or liver sample) from a NAFLDpatient: a) alanine aminotransferase (ALT); b) aspartateaminotransferase (AST); c) adiponectin; d) N-terminal fragment of typeIII collagen (proC3); e) caspase-cleaved keratin 18 fragments (M30 andM65); f) IL-1 beta; g) C-reactive protein; h) PIIINP; i) TIMP1; j)MCP-1; k) FGF-21; or 1) gamma glutamyl transferase (GGT). For example, asubject with NAFLD can exhibit a decrease in levels of one, two, or more(e.g., all) of ALT, AST, or GGT after treatment with the composition.

The patient may exhibit a mean change in plasma glucose, insulin,homeostatic model assessment insulin resistance (HOMA IR), lipidprofile, hemoglobin A1c (HbA1c) and other metabolic parameters from,e.g., baseline to Weeks 6 and 12, including changes in plasma glucoseand insulin levels in the setting of an oral glucose tolerance test(oGTT) from, e.g., baseline to Weeks 6 and 12. The patient may exhibit amean change in body weight from, e.g., baseline to Weeks 6 and 12.

Administration of the amino acid composition can result in animprovement in the NAFLD activity score, glucose tolerance, hepatocyteinflammation, liver fibrosis or liver injury, steatosis, or hepatocyteballooning in the patient.

Example 15 Treatment of NASH in a Mouse Model with an Amino AcidComposition

Induction of NASH in Mice

In one example, the effects of LIVRQNAC and related amino acidcompositions in the obesity, metabolism-driven non-alcoholicsteatohepatitis (NASH) in FATZO mouse model was examined.

Materials and Methods

Induction of NASH in Mice

NASH was induced in 60 male FATZO mice by a western diet (Research Diet# D12079B; fat 40% kcal, protein 17% kcal, carbohydrate 43% kcal)supplemented with 5% fructose in the drinking water (WDF) during a 16week induction phase. Diets and water were available ad libitum.Littermate control male FATZO mice fed with a control diet (n=6, Purina#5008; fat 17% kcal, protein 27% kcal, carbohydrate 56% kcal) andsterile water were set up for control purpose. Mice were housed inplastic cages with microisolator. Sterilized bedding was replaced once aweek. Mice were housed three per cage and maintained on a twelve hourlight cycle throughout study duration. Room temperature was monitoreddaily and maintained at 22-25° C. Body weight was recorded every weekduring the induction phase.

Study Design

Following 16 weeks diet induction, 6 mice remained on control diet(group 1, Control) while 60 induced mice were randomized on body weightand plasma glucose (fed) for assignment to the following treatments.FATZO mice were administered with test articles starting at 16 weekspost western diet NASH induction for 4 weeks. Test articles wereadministered by oral gavage. Animals were euthanized at 20 weeks postwestern diet NASH induction, and tissues were harvested for analysis.

Group (n) Treatment (oral) Diet 1 6 Vehicle 5008 WDF 2 10 VehicleD12079B + 5% Fructose 3 10 LIVRQNAC (1500 mg/kg) D12079B + 5% Fructose 410 LIVRQNAC (3000 mg/kg) D12079B + 5% Fructose 5 10 LIVRQNAC + G (3885mg/kg) D12079B + 5% Fructose 6 10 LRQNAC (2469 mg/kg) D12079B + 5%Fructose 7 10 Obeticholic acid (OCA) D12079B + 5% Fructose 30 mg/kg/dayTest Articles

LIVRQNAC, LIVRQNAC+G, LRQNAC, and OCA (Advanced ChemBlocks, Inc.),incipient, and water for irrigation were provided by Axcella Health,Inc. 0.5% Methylcellulosewas provided by CrownBio, Inc. Dosing solutionswere prepared according to Appendix 1. TA compounds (amino acidcompositions) were amino acid blends formulated fresh daily in water forirrigation (Baxter #27F7114) and the excipients 0.125% Xanthan Gum, 1.5mM Sodium Lauryl Sulfate and 0.28% Lecithin. Obeticholic acid (OCA) wassuspended in 0.5% methylcellulose in water for irrigation. All testarticles were stored refrigerated. TA compounds were provided in frozenpowder form by the sponsor. Dosing was continued for 4 weeks.

Leucine dosages of LIVRQNAC+G and LRQNAC were matched to that ofLIVRQNAC.

Amino Acid Compositions

Supplier Part Lot Ingredient Grade Supplier Number Number Fusi-BCAAInstantized Ajinomoto 33555 OH704 Unflavored (2:1:1 L- (0.3-0.9%(AjiPure) Leu:L-Ile:L-Val) Lecithin) L-Arginine HCl USP Sigma A4599CDB0352V (Ajinomoto) L-Arginine HCl USP Sigma A4599 CDB0352V (Ajinomoto)L-Glutamine USP Ajinomoto 32824 R014A003 Glycine USP Ajinomoto 30359R015T008 Acetylcysteine (NAC) USP Spectrum AC126 1FI0576 Chemical

LIVRQNAC LIVRQNAC + G LRQNAC Daily Daily Daily Ingredient Dose (g) Dose(g) Dose (g) Fusi-BCAA Unflavored 24.0 24.0 (2:1:1 L-Leu:L-Ile:L-Val)Fusil (L-Leucine) 12.0 L-Arginine HCl 18.0 18.0 18.0 L-Glutamine 24.024.0 24.0 Glycine 20.0 Acetylcysteine (NAC) 1.8 1.8 1.8 AMINO ACIDS =67.8 87.8 55.8Test Articles Administration

LIVRQNAC, LIVRQNAC+G, LRQNAC, OCA and Vehicle were administered by oralgavage at a volume of 10 mL/kg throughout the study. Dosages werecalculated by daily body weight. LIVRQNAC, LIVRQNAC+G, LRQNAC, andVehicle were administered twice per day (BID), while OCA wasadministered once a day (QD) in the morning. Mice receiving OCA once perday (QD), and one vehicle QD. Doses were administered by oral gavage at0700 and 1800 by oral gavage for 4 weeks.

Body Weight and Blood Glucose

The viability, clinical signs and behavior were monitored daily. Bodyweight was recorded daily during the dosing period. Blood samples werecollected weekly in the AM (0700) via tail clip for glucose measurement(StatStrip glucometer).

Necropsy and Sample Harvest

Animals were anesthetized with CO2 inhalation and exsanguinated viacardiac puncture for euthanasia. Terminal blood samples (K2EDTA) wereobtained by cardiac puncture in anesthetized animals at termination.Samples were provided frozen to Axcella Health. Organ weights (totalliver, gonadal fat pads) were recorded. Pancreas, and small intestineand gonadal fat pads were fixed in 10% Buffered Formalin and prepared asdirected in protocol. A section of small intestine, gonadal fat pad andliver were also snap frozen in liquid nitrogen and shipped to thesponsor.

Histological Analyses

The liver tissues were fixed in Bouin's solution at 4° C. for 24 hoursfollowed by baths of standard concentrations of alcohol then xylene toprepare the tissues for paraffin embedding. After being embedded inparaffin and cooled, five-micron sections were cut and stained forroutine H&E and Picric Sirius Red. A section of both right and leftlobes of the livers were frozen in OCT for analysis of lipid contentwith Oil-Red-) staining. The Aperio whole slide digital imaging system(Scan Scope CS, Vista, Calif.) was used for imaging. All slides wereimaged at 20×. The scan time ranged from 1.5 minutes to a maximum timeof 2.25 minutes. The whole images were housed and stored in theirSpectrum software system and images were shot from the whole slides.

The livers were evaluated using the NASH liver criteria for scoring. Inthis mouse study, one cross section of liver for each case was analyzedwith the NASH score system. According to the published NASH CRN ScoringSystem, this scoring system comprises of NAFLD Activity Score (NAS),fibrosis stage and identification of NASH by pattern recognition. TheNAS can range from 0 to 8 and is calculated by the sum of scores ofsteatosis (0-3), lobular inflammation (0-3) and hepatocyte ballooning(0-2) from H&E stained sections. Fibrosis was scored (0-4) frompicrosirius red stained slides. The NASH system is used for human liver18 gauge biopsies. Steatosis, lobular inflammation, hepatocyte. balloondegeneration, fibrosis, NAS and the presence of NASH by patternrecognition were systematically assessed. In this study we evaluated onetotal cross section of liver per mouse in this study. This is about 15times the size of an 18 gauge human liver biopsy. The pathology scorewas determined as 0, +1, +2, or +3. The lesions were scored on location(periportal, centrilobular, and mid zonal) and fat accumulation (focal,periportal, and/or centrilobular). The other part of the score wasdistribution of the lesions: focal, multifocal and/or diffuse. Also,mild, moderate and severity of the lesions. These parameters made up thetotal NASH score.

All immunohistochemical staining steps were performed using the DakoFLEX SYSTEM on an automated immunostainer; incubations were done at roomtemperature and Tris buffered saline plus 0.05% Tween 20, pH 7.4(TBS-Dako Corp.) was used for all washes and diluents. Thorough washingwas performed after each incubation. Primary antibodies includedanti-mouse SMA, F4/80, Mac-2, and Picric Sirius Red. Control sectionswere treated with an isotype control using the same concentration asprimary antibodies to verify the staining specificity.

White adipose tissue (WAT) adipocyte size was analyzed from the H&Estained sections. Using the Aperio Image Scope application, 3 localizedregions (edge of tissue, tissue not surrounding vascular area, tissuesurrounding vascular area) of each tissue specimen were assessed bymeasuring the area of 10 largest adipocytes of the region. Within eachtissue, 10 hot spots of each regions were quantified (um²) and averaged.

Pancreatic beta-islet cells were identified by immunohistochemicalstaining.

Image Analysis

Aperio Automatic Image Quantitation was employed to quantify positivepixels of immunohistochemical staining, Oil-Red 0, and Sirius Redstaining. The Positive Pixel Count algorithm was used to quantify thepercentage of a specific stain present in a scanned slide image. A rangeof color (range of hues and saturation) and three intensity ranges(weak, positive, and strong) were masked and evaluated. The algorithmcounted the number and intensity-sum in each intensity range, along withthree additional quantities: average intensity, ratio of strong/totalnumber, and average intensity of weak positive pixels. The positivepixel algorithm was modified to distinguish between the orange and bluecolors. Alterations from the normal “hue value” (0.1 to 0.96) and “colorsaturation” (0.04 to 0.29), were made for the Sirius Red evaluation.Vasculature and artifacts were excluded from analysis.

Liver Gene Expression Analysis

Liver gene expression of MCP-1 and MIP-1a was measured by quantitativePCR.

Liver Cytokine and Chemokine Measurement

Liver IL-1b, MCP-1, and MIP-1 protein levels were quantified using themultiplex ELISA Assay (Meso Scale Discovery, Rockville, Md.).

Statistical Analysis

Statistical analyses of liver histological scores were performed usingBonferroni Multiple Comparison Test on GraphPad Prism 6 (GraphPadSoftware Inc., USA). P values <0.05 were considered statisticallysignificant. Results were expressed as mean±SEM. Comparisons were madebetween Group 2 (Vehicle) and the following groups; Group 3 (LIVRQNAC1,500 mg/kg), Group 4 (LIVRQNAC 3,000 mg/kg), Group 5 (LIVRQNAC+G, 3,885mg/kg), and (LRQNAC, 2,469 mg/kg).

Results

Body and Liver Weight

Feeding the western diet supplemented with fructose (WDF) for 16 weekselicited significant effects on body weight compared to control fedanimals. Prior to administration of test agent, animals fed the WDF weresignificantly heavier (47.6±0.45 vs. 43.9±1.03 g; p<0.01) compared toanimals fed the control diet.

Body weight decreased compared to baseline values in all treatmentgroups; there were no significant differences in weight loss compared tovehicle (−7.6±0.9, −6.9±1.3, −6.8±1.4, −5.7±1.2, −6.4±1.0, −4.7±1.6 and−3.9±1.5% for control, vehicle, LIVRQNAC (1500 mg/kg), LIVRQNAC (3000mg/kg), LIVRQNAC+G, LRQNAC, and OCA, respectively; p<0.4992).

Liver weight (% body weight) was significantly higher in vehicle treatedanimals fed WDF compared to control diet (7.22±0.3 vs. 5.05±0.24%;p<0.0001); however, in animals fed WDF, no significant effects comparedto vehicle were noted in any treatment group (7.22±03, 7.14±0.3,7.19±0.26, 6.69±0.18, 7.02±0.5 and 6.81±0.2 for vehicle, LIVRQNAC (1500mg/kg), LIVRQNAC (3000 mg/kg), LIVRQNAC+G, LRQNAC, and OCA,respectively; p<0.7450).

Blood Glucose

Feeding the western diet supplemented with fructose (WDF) for 16 weekselicited significant effects on glycemia compared to control fedanimals. Prior to administration of test agent, animals fed the WDF hadsignificantly lower glucose (160.0±3.01 vs. 218.3±28.6 mg/dL; p<0.0001)compared to animals fed the control diet.

Blood glucose, although higher in control animals at baseline, remainedrelatively stable during 4 weeks of compound administration. Whenaveraged over the dosing period, there were no significant differencesin average blood glucose compared to vehicle for any treatment group(166.0±9.7, 157.1±4.6, 154.6±2.3, 159.4±3.8, 155.5±3.8, 153.6±3.0 and169.7±6.3 mg/dL for control, vehicle, LIVRQNAC (1500 mg/kg), LIVRQNAC(3000 mg/kg), LIVRQNAC+G, LRQNAC, and OCA, respectively; p<0.1587).

Liver Triglyceride and Cholesterol

Liver triglyceride and cholesterol content were similarly elevated afterWDF feeding compared to vehicle treated animals fed control diet (livertriglyceride p<0.0040; liver cholesterol: p<0.0001). Among animals fedWDF, there were no significant differences in liver triglyceride(p<0.1206) when compared to vehicle for any treatment group. While OCAreduced liver cholesterol content compared to vehicle by 32% (p<0.05),no amino acid composition treatment group affected liver cholesterol ascompared to WDF feeding vehicle group.

LIVRQNAC LIVRQNAC LIVRQNAC + Liver Vehicle 1.5 g/kg 3.0 g/kg G LRQNACOCA Triglyceride 31.49 ± 5.85  47.63 ± 1.19  47.94 ± 1.37  50.57 ± 1.58 49.47 ± 1.4  49.81 ± 1.63  Cholesterol  8.37 ± 0.065  7.74 ± 0.318  7.48± 0.697  6.42 ± 0.648  7.84 ± 0.104  5.63 ± 0.495Liver Histology

FATZO mice fed with the control diet developed mild steatosis and noinflammation, ballooning, or fibrosis (FIG. 10). FATZO mice fed with theWDF and treated with vehicle developed significant steatosis, mildinflammation, ballooning, and fibrosis. In contrast to predominantlymacrovesicular steatosis in the vehicle groups, a mixture ofpredominantly microvesicular and diminished macrovesicular steatosis wasobserved in LIVRQNAC, LIVRQNAC+G and LRQNAC groups, as shown in FIG. 11.

The NAFLD activity score is calculated from histological scoring ofsteatosis (0-3), inflammation (0-3), and ballooning (0-2) in fixed livertissues. In WDF fed animals, all amino acid composition treatmentsproduced a significant reduction in the NAS compared to the vehicletreatment group (FIG. 12). LIVRQNAC and amino acid compositiontreatments reduced liver steatosis as compared to vehicle, although onlyLIVRQNAC+G and LRQNAC reached statistical significance (p<0.05), whileLIVRQNAC did not (LIVRQNAC 3.0 g/kg, p=0.12). All amino acid compositiontreatments significantly attenuated hepatocyte ballooning, the biomarkerof lipotoxicity and cell death. Amino acid composition treatments didnot significantly alter liver inflammation. In conclusion, amino acidcomposition-associated improvement of liver pathology is mainlyattributed to attenuation of hepatocyte ballooning.

There was no significant effect of OCA on the NAS score and NAScomponents compared to vehicle.

Liver LIVRQNAC LIVRQNAC LIVRQNAC + Pathology Vehicle 1.5 g/kg 3.0 g/kg GLRQNAC OCA NAS  3.65 ± 0.183  2.70 ± 0.213  2.89 ± 0.111  2.83 ± 0.186 2.72 ± 0.147  3.72 ± 0.147 Steatosis  1.8 ± 0.133  1.6 ± 0.163  1.44 ±0.176  1.33 ± 0.167  1.33 ± 0.167  1.78 ± 0.147 Inflammation 0.9 ± 0.11.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 1.0 ± 0.0 Ballooning 0.95 ± 0.050.1 ± 0.1  0.44 ± 0.176  0.50 ± 0.144  0.39 ± 0.111  0.94 ± 0.056

Livers from vehicle treated animals demonstrated a mild fibrosis; scoreof 0.8±0.1. Only livers from animals treated with LIVRQNAC (1500 mg/kg)demonstrated a significant reduction in fibrosis when compared to thevehicle treated group, (0.2±0.1 versus 0.8±0.1, p<0.01), but not withLIVRQNAC (3000 mg/kg), LIVRQNAC+G or LRQNAC. Sirius Red collagenstaining demonstrated that all amino acid composition treatments hadsignificantly lower collagen deposition compared to vehicle (LIVRQNAC1500 mg/kg, p<0.01; LIVRQNAC 3000 mg/kg, p<0.01; LIVRQNAC+G, p=0.09;LRQNAC, p<0.05). OCA did not affect liver fibrosis score or Sirius Redcollagen staining area.

LIVRQNAC LIVRQNAC LIVRQNAC + Fibrosis Vehicle 1.5 g/kg 3.0 g/kg G LRQNACOCA Fibrosis  0.8 ± 0.133  0.2 ± 0.133  0.44 ± 0.176  0.44 ± 0.176  0.33± 0.167  0.67 ± 0.167 Sirius  1.82 ± 0.279  0.77 ± 0.116  0.72 ± 0.0920.107 ± 0.218  0.79 ± 0.183 1.59 ± 0.36 Red

Consistent with liver triglyceride levels, amino acid compositiontreatments did not alter liver Oil Red O staining area compared tovehicle group. OCA reduced Oil Red O staining area (p<0.05).

LIVRQNAC LIVRQNAC LIVRQNAC + Oil Red O Vehicle 1.5 g/kg 3.0 g/kg GLRQNAC OCA Oil Red O  0.32 ± 0.019  0.28 ± 0.022  0.30 ± 0.022  0.26 ±0.023  0.29 ± 0.018  0.24 ± 0.021 Triglyceride 31.49 ± 5.85  47.63 ±1.19  47.94 ± 1.37  50.57 ± 1.58  49.47 ± 1.4  49.81 ± 1.63 Liver Gene Expression

MCP-1 (CCL2) and MIP-1a (CCL3) are proinflammatory chemokines thatmediate liver inflammation via macrophage and neutrophil recruitment.MCP-1 and MIP-1a are the ligands of CCR2 and CCR5, respectively, whichserve the promising therapeutic targets to treat liver fibrosis in NASH.MCP-1 and MIP-1a RNA expression levels in the liver were significantlyupregulated in the WDF fed mice as compared to control diet-fed mice, asshown in Tables 74 and 75.

TABLE 74 Fold change in MCP-1 mRNA levels after administration of aminoacid compositions LIVRQNAC LRQNAC (3000 LIVRQNAC + G (2469 MCP-1 Ctrldiet Vehicle mg/kg) (3885 mg/kg) mg/kg) Mean 0.1457 1.079 1.396 0.61020.8777 SEM 0.0291 0.1956 0.3414 0.09597 0.2315

TABLE 75 Fold change in MIP-1a mRNA levels after administration of aminoacid compositions LIVRQNAC LRQNAC (3000 LIVRQNAC + G (2469 MIP-1a Ctrldiet Vehicle mg/kg) (3885 mg/kg) mg/kg) Mean 0.08328 1.194 1.67 0.8141.514 SEM 0.02141 0.25 0.3366 0.1029 0.525

LIVRQNAC and LRQNAC treatments did not significantly alter liver MCP-1and MIP-1a RNA expression as compared to vehicle group. LIVRQNAC+Gtreatment resulted in slightly lower liver MCP-1 RNA expression ascompared to vehicle group (p=0.054) and LIVRQNAC group (p<0.05).Similarly, LIVRQNAC+G treatment resulted in slightly lower liver MCP-1RNA expression as compared to vehicle group although the difference wasnot significant (p=0.19) and LIVRQNAC group (p<0.05).

Liver Chemokines and Cytokines

Consistent with RNA data, liver MCP-1 and MIP-1a protein levels wereelevated in the WDF fed mice as compared to control diet-fed mice, asshown in Tables 76 and 77.

TABLE 76 Mean liver MCP-1 protein levels after administration of aminoacid compositions LIVRQNAC LRQNAC (3000 LIVRQNAC + G (2469 MCP-1 Ctrldiet Vehicle mg/kg) (3885 mg/kg) mg/kg) Mean 41.47 278.3 392 221.1 247.1SEM 7.463 61.41 83.97 36.6 75.16

TABLE 77 Mean liver MIP-1a protein levels after administration of aminoacid compositions LIVRQNAC LRQNAC (3000 LIVRQNAC + G (2469 MIP-1a Ctrldiet Vehicle mg/kg) (3885 mg/kg) mg/kg) Mean 23.16 191.8 282.9 142.8141.1 SEM 5.429 30.03 58.88 17.04 24.36

Liver MCP-1 and MIP-1a protein levels were also positively correlatedwith RNA expression levels, as shown in Tables 78 and 79.

TABLE 78 Correlations between MCP-1 protein and RNA levels afteradministration of amino acid compositions Ctrl diet y = 0.0022x + 0.0542R2 = 0.3202 Vehicle y = 0.0029x + 0.3316 R2 = 0.7986 LIVRQNAC (3000mg/kg) y = 0.0036x + 0.0144 R2 = 0.7831 LIVRQNAC + G (3885 mg/kg) y =0.0018x + 0.2542 R2 = 0.3988 LRQNAC (2469 mg/kg) y = 0.0027x + 0.2969 R2= 0.6857

TABLE 79 Correlations between MIP-1a protein and RNA levels afteradministration of amino acid compositions Ctrl diet y = 0.001x + 0.0593R2 = .069 Vehicle y = 0.0057x + 0.191 R2 = 0.4202 LIVRQNAC (3000 mg/kg)y = 0.0051x + 0.2334 R2 = 0.7887 LIVRQNAC + G (3885 mg/kg) y = 0.0045x +0.1817 R2 = 0.4403 LRQNAC (2469 mg/kg) y = 0.0064x + 0.1814 R2 = 0.4875

LIVRQNAC and LRQNAC treatments did not significantly alter liver MCP-1and MIP-1a protein levels as compared to vehicle group. LIVRQNAC+Gtreatment slightly lowered liver MCP-1 (p=0.095) and MIP-1a (p<0.05)protein levels as compared to LIVRQNAC group. Additionally, liver MCP-1and MIP-1a protein levels positively correlated, as shown in Table 80.

TABLE 80 Correlations between MCP-1 and MIP-1a protein levels afteradministration of amino acid compositions Ctrl diet y = 0.6803x − 5.0537R2 = 0.8744 Vehicle y = 0.389x + 83.574 R2 = 0.6325 LIVRQNAC (3000mg/kg) y = 0.6615x + 23.609 R2 = 0.8903 LIVRQNAC + G (3885 mg/kg) y =0.4437x + 44.728 R2 = 0.9082 LRQNAC (2469 mg/kg) y = 0.3108x + 75.901 R2= 0.9241

Proinflammatory cytokines IL-1b, IL-6, TNFa, and CXCL1 protein levels inliver were elevated in the WDF fed mice as compared to control diet-fedmice, as shown in Tables 81-84.

TABLE 81 Mean liver IL-1b protein levels after administration of aminoacid compositions LIVRQNAC LRQNAC (3000 LIVRQNAC + G (2469 IL-1b Ctrldiet Vehicle mg/kg) (3885 mg/kg) mg/kg) Mean 10.53 22.31 22.11 14.4228.85 SEM 1.248 6.063 5.739 3.299 10.41

TABLE 82 Mean liver IL-6 protein levels after administration of aminoacid compositions LIVRQNAC LRQNAC (3000 LIVRQNAC + G (2469 IL-6 Ctrldiet Vehicle mg/kg) (3885 mg/kg) mg/kg) Mean 5.838 8.452 7.298 5.77 6.71SEM 0.3536 2.723 2.043 1.06 1.625

TABLE 83 Mean liver CXCL1 protein levels after administration of aminoacid compositions LIVRQNAC LRQNAC (3000 LIVRQNAC + G (2469 CXCL1 Ctrldiet Vehicle mg/kg) (3885 mg/kg) mg/kg) Mean 41.42 105.9 119.6 77.7784.9 SEM 2.487 13.26 26.15 8.023 10.49

TABLE 84 Mean liver TNFα protein levels after administration of aminoacid compositions LIVRQNAC LRQNAC (3000 LIVRQNAC + G (2469 TNFa Ctrldiet Vehicle mg/kg) (3885 mg/kg) mg/kg) Mean 1.703 3.71 4.574 2.9744.119 SEM 0.5641 0.4647 0.5654 0.1513 0.8341

LIVRQNAC, LIVRQNAC+G, and LRQNAC treatments did not significantly alterIL-1b, IL-6, TNFa, and CXCL1 protein levels as compared to vehicle.Liver TNFa levels were lower by LIVRQNAC+G treatment as compared toLIVRQNAC.

Summary

Based on clinical observations, WDF-fed FATZO mice gained more bodyweight that those fed with a control diet. Fed blood glucose levels werecomparable between WDF-fed and control diet-fed mice despite of thedifference in body weight change. All treatments were well tolerated inFATZO mice. Both WDF-fed and control diet-fed mice lose body weightduring the treatment period, which may be due to the stress associatedwith administration of test articles or vehicle via oral gavage twice aday.

NAS was significantly attenuated in all amino acid composition treatmentgroups as compared to vehicle, predominantly attributing to ballooningscore. Hepatocyte ballooning was significantly reduced in all the aminoacid composition treatment groups. Steatosis was significantly reducedin LIVRQNAC+G and LRQNAC treatment groups. LIVRQNAC also loweredsteatosis, although the difference was not significant Inflammation wasnot affected by amino acid composition treatments. Despite thehistological improvement in steatosis score in LIVRQNAC+G and LRQNACtreatment groups, liver triglyceride, cholesterol, and Oil-Red Ostaining remained unchanged by amino acid composition treatments.Consistent with the histological and biochemical data, de novolipogenesis enzymes FASN and ACACA RNA levels were not affected by aminoacid composition treatment.

Although liver triglyceride levels were not affected by amino acidcomposition treatments, the characteristics of hepatocyte steatosis werediffered by amino acid composition treatments. Liver of the WDF-fed mice(vehicle group) demonstrated predominantly macrovesicular steatosis. Incontrast, macrovesicular steatosis was diminished, and a mixture ofmicrovesicular and macrovesicular steatosis in all amino acidcomposition treatment groups. The biological meaning and mechanism ofamino acid compositions on macro- to microvesicular steatosis phenotypesmerit further investigation.

Liver fibrosis score in FATZO model of NAFLD was significantlyattenuated by LIVRQNAC treatment at low dose but not at high dose.LIVRQNAC+G and LRQNAC had no effect on fibrosis. Nonetheless, Sirius Redcollagen staining demonstrated that LIVRQNAC, LIVRQNAC+G and LRQNACsignificantly reduced collagen deposition in the liver.

Consistent with liver inflammation scores, liver RNA and protein levelsof the proinflammatory chemokine MCP-1 and MIP-1a and cytokines IL-1b,IL-6, TNFa, and CXCL1 were not significantly affected by amino acidcomposition treatment. It is of interest to note that LIVRQNAC+G(equivalent to LIVRQNAC plus Glycine) treatment had lower liver MCP-1,MIP-1a, and TNFa as compared to LIVRQNAC.

Increased liver oxidative stress associated with inflammation isobserved during NAFLD and NASH. Glutathione (GSH) is a pivotalendogenous anti-oxidant which can counteract reactive oxygen species.Glycine and its direct metabolic precursor, serine, are substrates forGSH biosynthesis. Thus, serine and/or glycine supplementation helpsreplenish GSH and ameliorates NAFLD and NASH. LIVRQNACG treatment hadlower inflammation chemokines and cytokines in the liver, supportingthat supplementation of glycine or serine is beneficial in NAFLD andNASH.

In conclusion, all three amino acid compositions (LIVRQNAC, LIVRQNAC+Gand LRQNAC) tested in FATZO mice attenuate NAFLD activity scores,hepatocyte ballooning, and fibrosis. These amino acid compositions canbe used to treat NASH. Glycine-containing amino acid compositions canfurther reduce liver inflammation which results in reduced liverfibrosis.

While the invention has been particularly shown and described withreference to a preferred embodiment and various alternate embodiments,it will be understood by persons skilled in the relevant art thatvarious changes in form and details can be made therein withoutdeparting from the spirit and scope of the invention.

All references, issued patents and patent applications cited within thebody of the instant specification are hereby incorporated by referencein their entirety, for all purposes.

What is claimed is:
 1. A composition comprising: (a) L-leucine or a saltthereof, or a dipeptide or salt thereof, or tripeptide or salt thereof,comprising L-leucine; (b) an arginine amino acid entity chosen from: (i)L-arginine or a salt thereof, (ii) a dipeptide or salt thereof, ortripeptide or salt thereof, comprising L-arginine, (iii) ornithine or asalt thereof, (iv) a dipeptide or salt thereof, or tripeptide or saltthereof, comprising ornithine, (v) creatine or a salt thereof, or (vi) adipeptide or salt thereof, or tripeptide or salt thereof, comprisingcreatine; (c) L-glutamine or a salt thereof or a dipeptide or saltthereof, or tripeptide or salt thereof, comprising L-glutamine; and (d)N-acetylcysteine (NAC) or a salt thereof or a dipeptide or salt thereof,or tripeptide or salt thereof, comprising NAC; wherein when thecomposition is in powder form, at least 50 wt. % of the total wt. of thecomposition is one or more amino acid entities in free form, and whereinthe total wt. % of (a)-(d) is greater than the total wt. % of otheramino acid entities in the composition.
 2. The composition of claim 1,wherein methionine, tryptophan, and cysteine are present at less than 2wt. % of the composition when the composition is in powder form.
 3. Thecomposition of claim 1, wherein methionine, tryptophan, or cysteine areabsent in the composition.
 4. The composition of claim 1, wherein: (1)the wt. % of the L-glutamine or a salt thereof or the dipeptide or saltthereof, or tripeptide or salt thereof, comprising L-glutamine in thecomposition is greater than the wt. % of the arginine amino acid entity;(2) the wt. % of the L-glutamine or a salt thereof or the dipeptide orsalt thereof, or tripeptide or salt thereof, comprising L-glutamine inthe composition is equal to or greater than the wt. % of the leucine ora salt thereof, or a dipeptide or salt thereof, or tripeptide or saltthereof, comprising L-leucine; or (3) the wt. % of the arginine aminoacid entity in the composition is greater than the wt. % of theL-leucine or salt thereof, or a dipeptide or salt thereof, or tripeptideor salt thereof, comprising L-leucine.
 5. The composition of claim 1,wherein: the wt. % of the arginine amino acid entity in (b), theL-glutamine or a salt thereof or the dipeptide or salt thereof, ortripeptide or salt thereof, comprising L-glutamine in (c), and the NACor a salt thereof or the dipeptide or salt thereof, or tripeptide orsalt thereof, comprising NAC in (d) in combination is at least 50% ofthe composition, when the composition is in powder form, but not morethan 90% of the composition, when the composition is in powder form. 6.The composition of claim 1, wherein: (1) the ratio of the leucine or asalt thereof, or a dipeptide or salt thereof, or tripeptide or saltthereof, comprising L-leucine to the arginine amino acid entity is atleast 1:4, and not more than 3:4; (2) the ratio of the leucine leucineor a salt thereof, or a dipeptide or salt thereof, or tripeptide or saltthereof, comprising L-leucine to the L-glutamine or a salt thereof orthe dipeptide or salt thereof, or tripeptide or salt thereof, comprisingL-glutamine is at least 1:4, and not more than 3:4; or (3) the ratio ofthe arginine amino acid entity to the L-glutamine or a salt thereof orthe dipeptide or salt thereof, or tripeptide or salt thereof, comprisingL-glutamine is at least 1:4, and not more than 6:7.
 7. The compositionof claim 1, wherein the wt. ratio of: (a) leucine or a salt thereof, ora dipeptide or salt thereof, or tripeptide or salt thereof, comprisingL-leucine, (b) the arginine amino acid entity, (c) the L-glutamine or asalt thereof or the dipeptide or salt thereof, or tripeptide or saltthereof, comprising L-glutamine, and (d) the NAC or salt thereof or thedipeptide or salt thereof, or tripeptide or salt thereof, comprising NACis, respectively, 1+/−15%:1.5+/−15%:2+/−15%:0.15+/−15%;1+/−15%:1.5+/−15%:2+/−15%:0.3+/−15%; or1+/−15%:0.75+/−15%:1+/−15%:0.225+/−15%.
 8. The composition of claim 1,further comprising one or both of: (1) L-isoleucine or a salt thereof ora dipeptide or salt thereof, or tripeptide or salt thereof, comprisingL-isoleucine; or (2) L-valine or a salt thereof or a dipeptide or saltthereof, or tripeptide or salt thereof, comprising L-valine.
 9. Thecomposition of claim 8, wherein the composition comprises: (a) L-leucineor a salt thereof; (b) L-arginine or a salt thereof; (c) L-glutamine ora salt thereof; (d) NAC or a salt thereof; (e) L-isoleucine or a saltthereof; and (f) L-valine or a salt thereof.
 10. The composition ofclaim 9, wherein the composition comprises: (a) L-leucine or a saltthereof; (b) L-arginine or a salt thereof; (c) L-glutamine or a saltthereof; (d) NAC or a salt thereof; (e) L-isoleucine or a salt thereof;and (f) L-valine or a salt thereof; wherein the wt. ratio of theL-leucine or a salt thereof, the L-isoleucine or a salt thereof, theL-valine or a salt thereof, the L-arginine or a salt thereof, theL-glutamine or a salt thereof, and the NAC or salt thereof is,respectively, 1+/−15%:0.5+/−15%:0.5+/−15%:1.5+/−15%:2+/−15%:0.15+/−15%or 1+/−15%:0.5+/−15%:0.5+/−15%:1.5+/−15%:2+/−15%:0.3+/−15%.
 11. Thecomposition of claim 1, wherein at least one of (a)-(d) is a free aminoacid.
 12. The composition of claim 1, wherein at least one of (a)-(d) isin a salt form.
 13. The composition of claim 1, wherein the compositioncomprises a combination of not more than 15 different amino acidentities.
 14. The composition of claim 1, further comprising a serineamino acid entity chosen from L-serine, L-glycine, or a combinationthereof.
 15. The composition of claim 1, wherein: the arginine aminoacid entity is chosen from: i) L-arginine or a salt thereof, ii) adipeptide or salt thereof, or tripeptide or salt thereof, comprisingL-arginine, iii) ornithine or a salt thereof, or iv) a dipeptide or saltthereof, or tripeptide or salt thereof, comprising ornithine.
 16. Thecomposition of claim 1, wherein the wt. % of the NAC or a salt thereofor the dipeptide or salt thereof, or tripeptide or salt thereof,comprising NAC is at least 1%, but not more than 10% or the composition,when the composition is in powder form.
 17. The composition of claim 1,further comprising L-serine or a salt thereof.
 18. A dietary compositioncomprising the composition of claim
 1. 19. A pharmaceutical compositioncomprising the composition of claim
 1. 20. A pharmaceutical compositionconsisting essentially of: (a) L-leucine or a salt thereof; (b)L-arginine or a salt thereof; (c) L-glutamine or a salt thereof; (d) NACor a salt thereof; (e) L-isoleucine or a salt thereof; (f) L-valine or asalt thereof; and (g) one or more pharmaceutically acceptableexcipients.